Organic compositions to treat Beta-ENaC-related diseases

ABSTRACT

The present disclosure relates to RNAi agents useful in methods of treating Beta-ENaC-related diseases such as cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle&#39;s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension, using a therapeutically effective amount of a RNAi agent to Beta-ENaC.

This application is a divisional application of U.S. Utility patentapplication Ser. No. 13/090,580 filed 20 Apr. 2011, which claimspriority to U.S. Provisional Application Ser. No. 61/333,398 filed 11May 2010 and U.S. Provisional Application Ser. No. 61/327,379 filed 23Apr. 2010, the contents of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

The mucosal surface between the environment and the body has manyprotective mechanisms. One form of defense is cleansing the surface withliquid. The quantity of liquid reflects the balance between epithelialliquid secretion (which often reflects anion secretion coupled withwater and a cation counter-ion) and liquid absorption (which oftenreflects Na⁺ absorption, coupled with water and counter anion). Manydiseases of mucosal surfaces are caused by too little liquid, as causedby an imbalance between secretion (too little) and absorption (toomuch). One method to balance the liquid layer is to decrease Na⁺channel-mediated liquid absorption.

Nonvoltage-gated, amiloride-sensitive sodium channels control fluid andelectrolyte transport across epithelia in many organs. The apicalmembranes of many tight epithelia contain sodium channels that areprimarily characterized by their high affinity to the diuretic blockeramiloride. These channels mediate the first step of active sodiumreabsorption essential for the maintenance of body salt and waterhomeostasis. In vertebrates, the channels control reabsorption of sodiumin the kidney, colon, lung and sweat glands; they also play a role intaste perception.

The rate-limiting step of Na⁺ and liquid absorption is mediated by theepithelial sodium (Na⁺) channel (ENaC). These sodium channels areheteromeric complexes consisting of 3 subunits: Alpha-ENaC, Beta-ENaC,and Gamma-ENaC.

Beta-ENaC (also known as SCNN1B) encodes the beta subunit of this sodiumchannel, and mutations in and/or altered expression of this gene havebeen associated with several diseases (and/or associated with treatmentsof diseases), including cystic fibrosis, pseudohypoaldosteronism type 1(PHA1), Liddle's syndrome, hypertension, alkalosis, hypokalemia, andobesity-associated hypertension.

There exists the need for treatments related to Beta-ENaC-relateddiseases.

BRIEF SUMMARY OF THE INVENTION

The present disclosure encompasses RNAi agents to Beta-ENaC, which areuseful in the treatment of Beta-ENaC-related diseases, such as cysticfibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome,hypertension, alkalosis, hypokalemia, and obesity-associatedhypertension. The present disclosure also encompasses a method oftreating a human subject having a pathological state mediated at leastin part by alpha-ENaC expression, the method comprising the step ofadministering to the subject a therapeutically effective amount of aRNAi agent Beta-ENaC.

The present disclosure provides specific RNAi agents and methods thatare useful in reducing Beta-ENaC levels in a subject, e.g., a mammal,such as a human. The present disclosure specifically providesdouble-stranded RNAi agents comprising at least 15 or more contiguousnucleotides of Beta-ENaC. In particular, the present disclosure providesagents comprising sequences of 15 or more contiguous nucleotidesdiffering by 0, 1, 2 or 3 from those of the RNAi agents provided, e.g.,in Table 1. The RNAi agents particularly can in one embodiment compriseless than 30 nucleotides per strand, e.g., such as 18-23 nucleotides,and/or 19-21 nucleotides, and/or such as those provided, e.g., in Table1.

The double-stranded RNAi agents can have blunt ends or overhangs of 1,2, 3 or 4 nucleotides (i.e., 1-4 nt) from one or both 3′ and/or 5′ ends.The double-stranded RNAi agents can also optionally comprise one or two3′ caps and/or one or more modified nucleotides. Modified variants ofsequences as provided herein include those that are otherwise identicalbut contain substitutions of a naturally occurring nucleotide for acorresponding modified nucleotide.

Further, the RNAi agent can either contain only naturally-occurringribonucleotide subunits, or one or more modifications to the sugar,phosphate or base of one or more of the replacement nucleotide subunits,whether they comprise ribonucleotide subunits or deoxyribonucleotidesubunits. In one embodiment, modified variants of the disclosed RNAiagents include RNAi agents with the same sequence, but with one or moremodifications to one or more of the sugar, phosphate or base of one ormore of the nucleotide subunits. In one embodiment, the modificationsimprove efficacy, stability and/or reduce immunogenicity of the RNAiagent. One aspect of the present disclosure relates to a double-strandedoligonucleotide comprising at least one non-natural nucleobase. Incertain embodiments, the non-natural nucleobase is difluorotolyl,nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particularembodiment, the non-natural nucleobase is difluorotolyl. In certainembodiments, only one of the two oligonucleotide strands contains anon-natural nucleobase. In certain embodiments, both of theoligonucleotide strands contain a non-natural nucleobase.

The RNAi agent(s) can optionally be attached to a ligand selected toimprove one or more characteristic, such as, e.g., stability,distribution and/or cellular uptake of the agent, e.g., cholesterol or aderivative thereof. The RNAi agent(s) can be isolated or be part of apharmaceutical composition used for the methods described herein.Particularly, the pharmaceutical composition can be formulated fordelivery to the lungs or nasal passage or formulated for parentaladministration. The pharmaceutical compositions can optionally comprisetwo or more RNAi agents, each one directed to the same or a differentsegment of the Beta-ENaC mRNA. Optionally, the pharmaceuticalcompositions can further comprise or be used in conjunction with anyknown treatment for any Beta-ENaC-related disease.

The present disclosure further provides methods for reducing the levelof Beta-ENaC mRNA in a cell, particularly in the case of a diseasecharacterized by over-expression or hyper-activity of ENaC. The presentdisclosure also encompasses a method of treating a human subject havinga pathological state mediated at least in part by Beta-ENaC expression,the method comprising the step of administering to the subject atherapeutically effective amount of a RNAi agent Beta-ENaC. Such methodscomprise the step of administering one of the RNAi agents of the presentdisclosure to a subject, as further described below. The present methodsutilize the cellular mechanisms involved in RNA interference toselectively degrade the target RNA in a cell and are comprised of thestep of contacting a cell with one of the RNAi agents of the presentdisclosure. Such methods can be performed directly on a cell or can beperformed on a mammalian subject by administering to a subject one ofthe RNAi agents/pharmaceutical compositions of the present disclosure.Reduction of target Beta-ENaC RNA in a cell results in a reduction inthe amount of encoded Beta-ENaC protein produced. In an organism, thiscan result in reduction of epithelial potential difference, balancedfluid absorption and increased mucociliary clearance.

The methods and compositions of the present disclosure, e.g., themethods and Beta-ENaC RNAi agent compositions, can be used with anydosage and/or formulation described herein, as well as with any route ofadministration described herein.

The details of one or more embodiments of the present disclosure are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the present disclosure will beapparent from this description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict the ability of RNAi agents AD20807, AD20826, AD20832,AS20834, AD20848, and AD20861 to knock-down Beta-ENaC activity in vivo.

FIGS. 2A-2C depict the in vitro effect of Beta-ENaC RNAi Agent AD20832on ENaC channel functional activity in human bronchial epithelial cells.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure encompasses RNAi agents to Beta-ENaC, which areuseful in treatment of Beta-ENaC-related diseases (e.g., diseasesassociated with mutations in and/or altered expression, level and/oractivity of Beta-ENaC, and/or diseases treatable by modulating theexpression, level and/or activity of Beta-ENaC), such as cysticfibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome,hypertension, alkalosis, hypokalemia, and obesity-associatedhypertension. The present disclosure also provides methods of treating ahuman subject having a pathological state mediated at least in part byBeta-ENaC expression, the method comprising the step of administering tothe subject a therapeutically effective amount of a RNAi agentBeta-ENaC.

Various Embodiments of the Present Disclosure Include:

A RNAi Agent Comprising an Antisense Strand Described Herein.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of a RNAi agent specific to Beta-ENaC (or any set of overlappingRNAi agents specific to Beta-ENaC) provided, e.g., in Table 1. Inanother embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense and an anti-sense strand,wherein the anti-sense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sensestrand of a RNAi agent from any sequence provided herein. In anotherembodiment, the present disclosure relates to a composition comprising aRNAi agent comprising a first strand and a second strand, wherein thefirst strand comprises at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nucleotides from the sequence of the first strand, and thesecond strand comprises at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nucleotides from the sequence of the second strand of anyRNAi agent provided herein.

Particular duplexes include the following, wherein each duplex comprisesa set of SEQ ID NOs, wherein the first SEQ ID NO corresponds to a firststrand (e.g., a sense strand), and the second SEQ ID NO corresponds to asecond strand (e.g., an anti-sense strand): AD-20805 (SEQ ID NOs. 111and 112); AD-20806 (SEQ ID NOs. 113 and 114); AD-20807 (SEQ ID NOs. 115and 116); AD-20808 (SEQ ID NOs. 117 and 118); AD-20809 (SEQ ID NOs. 119and 120); AD-20810 (SEQ ID NOs. 121 and 122); AD-20811 (SEQ ID NOs. 123and 124); AD-20812 (SEQ ID NOs. 125 and 126); AD-20813 (SEQ ID NOs. 127and 128); AD-20814 (SEQ ID NOs. 129 and 130); AD-20815 (SEQ ID NOs. 131and 132); AD-20816 (SEQ ID NOs. 133 and 134); AD-20817 (SEQ ID NOs. 135and 136); AD-20818 (SEQ ID NOs. 137 and 138); AD-20819 (SEQ ID NOs. 139and 140); AD-20820 (SEQ ID NOs. 141 and 142); AD-20821 (SEQ ID NOs. 143and 144); AD-20822 (SEQ ID NOs. 145 and 146); AD-20823 (SEQ ID NOs. 147and 148); AD-20824 (SEQ ID NOs. 149 and 150); AD-20825 (SEQ ID NOs. 151and 152); AD-20826 (SEQ ID NOs. 153 and 154); AD-20827 (SEQ ID NOs. 155and 156); AD-20828 (SEQ ID NOs. 157 and 158); AD-20829 (SEQ ID NOs. 159and 160); AD-20830 (SEQ ID NOs. 161 and 162); AD-20831 (SEQ ID NOs. 163and 164); AD-20832 (SEQ ID NOs. 165 and 166); AD-20833 (SEQ ID NOs. 167and 168); AD-20834 (SEQ ID NOs. 169 and 170); AD-20835 (SEQ ID NOs. 171and 172); AD-20836 (SEQ ID NOs. 173 and 174); AD-20837 (SEQ ID NOs. 175and 176); AD-20838 (SEQ ID NOs. 177 and 178); AD-20839 (SEQ ID NOs. 179and 180); AD-20840 (SEQ ID NOs. 181 and 182); AD-20841 (SEQ ID NOs. 183and 184); AD-20842 (SEQ ID NOs. 185 and 186); AD-20843 (SEQ ID NOs. 187and 188); AD-20844 (SEQ ID NOs. 189 and 190); AD-20845 (SEQ ID NOs. 191and 192); AD-20846 (SEQ ID NOs. 193 and 194); AD-20847 (SEQ ID NOs. 195and 196); AD-20848 (SEQ ID NOs. 197 and 198); AD-20849 (SEQ ID NOs. 199and 200); AD-20850 (SEQ ID NOs. 201 and 202); AD-20851 (SEQ ID NOs. 203and 204); AD-20852 (SEQ ID NOs. 205 and 206); AD-20861 (SEQ ID NOs. 207and 208); AD-20862 (SEQ ID NOs. 209 and 210); AD-20863 (SEQ ID NOs. 211and 212); AD-20864 (SEQ ID NOs. 213 and 214); AD-20865 (SEQ ID NOs. 215and 216); AD-20866 (SEQ ID NOs. 217 and 218); and AD-20867 (SEQ ID NOs.219 and 220), and modified variants thereof.

One embodiment provides modified variants of particular duplexes,wherein each duplex comprises a set of SEQ ID NOs, wherein the first SEQID NO corresponds to a first strand (e.g., a sense strand), and thesecond SEQ ID NO corresponds to a second strand (e.g., an anti-sensestrand) that are selected from the group consisting of: AD-20805 (SEQ IDNOs. 1 and 2); AD-20806 (SEQ ID NOs. 3 and 4); AD-20807 (SEQ ID NOs. 5and 6); AD-20808 (SEQ ID NOs. 7 and 8); AD-20809 (SEQ ID NOs. 9 and 10);AD-20810 (SEQ ID NOs. 11 and 12); AD-20811 (SEQ ID NOs. 13 and 14);AD-20812 (SEQ ID NOs. 15 and 16); AD-20813 (SEQ ID NOs. 17 and 18);AD-20814 (SEQ ID NOs. 19 and 20); AD-20815 (SEQ ID NOs. 21 and 22);AD-20816 (SEQ ID NOs. 23 and 24); AD-20817 (SEQ ID NOs. 25 and 26);AD-20818 (SEQ ID NOs. 27 and 28); AD-20819 (SEQ ID NOs. 29 and 30);AD-20820 (SEQ ID NOs. 31 and 32); AD-20821 (SEQ ID NOs. 33 and 34);AD-20822 (SEQ ID NOs. 35 and 36); AD-20823 (SEQ ID NOs. 37 and 38);AD-20824 (SEQ ID NOs. 39 and 40); AD-20825 (SEQ ID NOs. 41 and 42);AD-20826 (SEQ ID NOs. 43 and 44); AD-20827 (SEQ ID NOs. 45 and 46);AD-20828 (SEQ ID NOs. 47 and 48); AD-20829 (SEQ ID NOs. 49 and 50);AD-20830 (SEQ ID NOs. 51 and 52); AD-20831 (SEQ ID NOs. 53 and 54);AD-20832 (SEQ ID NOs. 55 and 56); AD-20833 (SEQ ID NOs. 57 and 58);AD-20834 (SEQ ID NOs. 59 and 60); AD-20835 (SEQ ID NOs. 61 and 62);AD-20836 (SEQ ID NOs. 63 and 64); AD-20837 (SEQ ID NOs. 65 and 66);AD-20838 (SEQ ID NOs. 67 and 68); AD-20839 (SEQ ID NOs. 69 and 70);AD-20840 (SEQ ID NOs. 71 and 72); AD-20841 (SEQ ID NOs. 73 and 74);AD-20842 (SEQ ID NOs. 75 and 76); AD-20843 (SEQ ID NOs. 77 and 78);AD-20844 (SEQ ID NOs. 79 and 80); AD-20845 (SEQ ID NOs. 81 and 82);AD-20846 (SEQ ID NOs. 83 and 84); AD-20847 (SEQ ID NOs. 85 and 86);AD-20848 (SEQ ID NOs. 87 and 88); AD-20849 (SEQ ID NOs. 89 and 90);AD-20850 (SEQ ID NOs. 91 and 92); AD-20851 (SEQ ID NOs. 93 and 94);AD-20852 (SEQ ID NOs. 95 and 96); AD-20861 (SEQ ID NOs. 97 and 98);AD-20862 (SEQ ID NOs. 99 and 100); AD-20863 (SEQ ID NOs. 101 and 102);AD-20864 (SEQ ID NOs. 103 and 104); AD-20865 (SEQ ID NOs. 105 and 106);AD-20866 (SEQ ID NOs. 107 and 108); and AD-20867 (SEQ ID NOs. 109 and110).

Particular Compositions

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising an antisense strand, wherein theantisense strand comprises at least 15 contiguous nucleotides differingby 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agentto Beta-ENaC selected from any sequence (or overlapping set ofsequences) provided in a table here (e.g., Table 1). In one embodiment,the present disclosure relates to a composition comprising a RNAi agentcomprising a sense strand and an antisense strand, wherein the antisensestrand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the antisense strand of a RNAi agent toBeta-ENaC selected from any sequence (or overlapping set of sequences)provided in a table here (e.g., Table 1). In another embodiment, thepresent disclosure relates to a composition comprising a RNAi agentcomprising a sense and an anti-sense strand, wherein the anti-sensestrand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the anti-sense strand of a RNAi agent from anysequence provided herein. In another embodiment, the present disclosurerelates to a composition comprising a RNAi agent comprising a firststrand and a second strand, wherein the first strand comprises at least15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides fromthe sequence of the first strand, and the second strand comprises atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotidesfrom the sequence of the second strand of any RNAi agent providedherein. Particular duplexes include those specific duplexes providedabove and as listed in any one or more of Table 1. Additional modifiedsequences (e.g., sequences comprising one or more modified base) of eachof the compositions above are also contemplated as part of the presentdisclosure.

Table A1, below, provides the SEQ ID NOs for the unmodified and anexample modified sequence of the sense and an anti-sense strands ofvarious RNAi agents to Beta-ENaC. The base composition of each isspecific sequence represented by the SEQ ID NOs is provided in moredetail in Table 1, and portions thereof are provided in Table 2.

TABLE A1 SEQ ID NOs for a first and a second strand (e.g., sense (“SS”)and anti- sense (“AS”) strand) for RNAi agents to Beta-ENaC ModifiedUnmodified RNAi agent- sequence sequence duplex name SEQ ID NO SEQ ID NOAD-20805 Sense 1 111 Anti-Sense 2 112 AD-20806 Sense 3 113 Anti-Sense 4114 AD-20807 Sense 5 115 Anti-Sense 6 116 AD-20808 Sense 7 117Anti-Sense 8 118 AD-20809 Sense 9 119 Anti-Sense 10 120 AD-20810 Sense11 121 Anti-Sense 12 122 AD-20811 Sense 13 123 Anti-Sense 14 124AD-20812 Sense 15 125 Anti-Sense 16 126 AD-20813 Sense 17 127 Anti-Sense18 128 AD-20814 Sense 19 129 Anti-Sense 20 130 AD-20815 Sense 21 131Anti-Sense 22 132 AD-20816 Sense 23 133 Anti-Sense 24 134 AD-20817 Sense25 135 Anti-Sense 26 136 AD-20818 Sense 27 137 Anti-Sense 28 138AD-20819 Sense 29 139 Anti-Sense 30 140 AD-20820 Sense 31 141 Anti-Sense32 142 AD-20821 Sense 33 143 Anti-Sense 34 144 AD-20822 Sense 35 145Anti-Sense 36 146 AD-20823 Sense 37 147 Anti-Sense 38 148 AD-20824 Sense39 149 Anti-Sense 40 150 AD-20825 Sense 41 151 Anti-Sense 42 152AD-20826 Sense 43 153 Anti-Sense 44 154 AD-20827 Sense 45 155 Anti-Sense46 156 AD-20828 Sense 47 157 Anti-Sense 48 158 AD-20829 Sense 49 159Anti-Sense 50 160 AD-20830 Sense 51 161 Anti-Sense 52 162 AD-20831 Sense53 163 Anti-Sense 54 164 AD-20832 Sense 55 165 Anti-Sense 56 166AD-20833 Sense 57 167 Anti-Sense 58 168 AD-20834 Sense 59 169 Anti-Sense60 170 AD-20835 Sense 61 171 Anti-Sense 62 172 AD-20836 Sense 63 173Anti-Sense 64 174 AD-20837 Sense 65 175 Anti-Sense 66 176 AD-20838 Sense67 177 Anti-Sense 68 178 AD-20839 Sense 69 179 Anti-Sense 70 180AD-20840 Sense 71 181 Anti-Sense 72 182 AD-20841 Sense 73 183 Anti-Sense74 184 AD-20842 Sense 75 185 Anti-Sense 76 186 AD-20843 Sense 77 187Anti-Sense 78 188 AD-20844 Sense 79 189 Anti-Sense 80 190 AD-20845 Sense81 191 Anti-Sense 82 192 AD-20846 Sense 83 193 Anti-Sense 84 194AD-20847 Sense 85 195 Anti-Sense 86 196 AD-20848 Sense 87 197 Anti-Sense88 198 AD-20849 Sense 89 199 Anti-Sense 90 200 AD-20850 Sense 91 201Anti-Sense 92 202 AD-20851 Sense 93 203 Anti-Sense 94 204 AD-20852 Sense95 205 Anti-Sense 96 206 AD-20861 Sense 97 207 Anti-Sense 98 208AD-20862 Sense 99 209 Anti-Sense 100 210 AD-20863 Sense 101 211Anti-Sense 102 212 AD-20864 Sense 103 213 Anti-Sense 104 214 AD-20865Sense 105 215 Anti-Sense 106 216 AD-20866 Sense 107 217 Anti-Sense 108218 AD-20867 Sense 109 219 Anti-Sense 110 220

For example, in Table A1, an exemplary modified sequence of RNAi agentAD-20805 is represented by SEQ ID NO: 1 (the sense strand) and SEQ IDNO: 2 (the anti-sense strand). The unmodified sequence of AD-20805 isrepresented by SEQ ID NO: 111 (the sense strand) and SEQ ID NO: 112 (theanti-sense strand). Thus, Table A1 presents the SEQ ID NO identifiers ofa first and second strand of the unmodified sequence and at least oneexemplary modified sequence for each of the various RNAi agents toBeta-ENaC.

An RNAi Agent Comprising an Anti-Sense Strand Described Herein

In one particular specific embodiment, the present disclosure relates toa composition comprising a RNAi agent comprising an anti-sense strand,wherein the anti-sense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sensestrand of a RNAi agent to Beta-ENaC selected from those anti-sensestrands in the specific duplexes provided herein and as listed, e.g., inTable 1.

Various specific embodiments of this embodiment are described below.

In one embodiment, the composition further comprises a second RNAi agentto Beta-ENaC. In various embodiments, the second RNAi agent isphysically separate from the first, or the two are physically connected(e.g., covalently linked or otherwise conjugated).

In one embodiment, the antisense strand is about 30 or fewer nt inlength.

In one embodiment, the sense strand and the antisense strand form aduplex region of about 15 to about 30 nucleotide pairs in length.

In one embodiment, the antisense strand is about 15 to about 36 nt inlength, including about 18 to about 30 nt in length, and furtherincluding about 19 to about 23 nt in length. In one embodiment, theantisense strand has at least the length selected from about 15 nt,about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt,about 27 nt, about 28 nt, about 29 nt and about 30 nt.

In one embodiment, the RNAi agent comprises a modification that causesthe RNAi agent to have increased stability in a biological sample orenvironment, e.g., blood serum or intestinal lavage fluid.

In one embodiment, the RNAi agent comprises at least one sugar backbonemodification (e.g., phosphorothioate linkage) and/or at least one2′-modified nucleotide. In one embodiment, all the pyrimidines are 2′O-methyl-modified nucleotides.

In one embodiment, the RNAi agent comprises: at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; and/or at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; and/or at least one 5′-cytidine-adenine-3′ (5′-ca-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide;and/or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide,wherein the 5′-uridine is a 2′-modified nucleotide.

In one embodiment, the RNAi agent comprises a 2′-modification selectedfrom the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA). In one embodiment, all thepyrimidines are 2′ O-methyl-modified nucleotides.

In one embodiment, the RNAi agent comprises a blunt end.

In one embodiment, the RNAi agent comprises an overhang having 1 to 4unpaired nucleotides.

In one embodiment, the RNAi agent comprises an overhang at the 3′-end ofthe antisense strand of the RNAi agent.

In one embodiment, the RNAi agent is ligated to one or more diagnosticcompound, reporter group, cross-linking agent, nuclease-resistanceconferring moiety, natural or unusual nucleobase, lipophilic molecule,cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin,terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 60% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 70% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 80% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 90% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi has an EC50 of no more than about 0.1 nM.

In one embodiment, the RNAi has an EC50 of no more than about 0.01 nM.

In one embodiment, the RNAi has an EC50 of no more than about 0.001 nM.

A RNAi Agent Comprising a First and a Second Strand Described Herein

In one particular specific embodiment, the present disclosure relates toa composition comprising a RNAi agent comprising a first strand and asecond strand, wherein the first strand and second strand comprise atleast 15 contiguous nucleotides, each differing by 0, 1, 2, or 3nucleotides from the first and second strand, respectively, of a RNAiagent to Beta-ENaC selected from the specific duplexes provided hereinand listed, e.g., in Table 1.

Various specific embodiments of this embodiment are described below.

In one embodiment, the composition further comprises a second RNAi agentto Beta-ENaC. In various embodiments, the second RNAi agent isphysically separate from the first, or the two are physically connected(e.g., covalently linked or otherwise conjugated).

In one embodiment, the antisense strand is about 30 or fewer nt inlength.

In one embodiment, the sense strand and the antisense strand form aduplex region of about 15 to about 30 nt pairs in length.

In one embodiment, the antisense strand is about 15 to about 36 nt inlength, including about 18 to about 23 nt in length, and including about19 to about 23 nt in length.

In one embodiment, the antisense strand has at least the length selectedfrom about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt,about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt and about 30nt.

In one embodiment, the RNAi agent comprises a modification that causesthe RNAi agent to have increased stability in a biological sample orenvironment, e.g., blood serum or intestinal lavage fluid.

In one embodiment, the RNAi agent comprises at least one sugar backbonemodification (e.g., phosphorothioate linkage) and/or at least one2′-modified nucleotide. In one embodiment, all the pyrimidines are 2′O-methyl-modified nucleotides.

In one embodiment, the RNAi agent comprises: at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; and/or at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; and/or at least one 5′-cytidine-adenine-3′ (5′-ca-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide;and/or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide,wherein the 5′-uridine is a 2′-modified nucleotide.

In one embodiment, the RNAi agent comprises a 2′-modification selectedfrom the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA).

In one embodiment, the RNAi agent comprises a blunt end.

In one embodiment, the RNAi agent comprises an overhang having 1 to 4unpaired nucleotides.

In one embodiment, the RNAi agent comprises an overhang at the 3′-end ofthe antisense strand of the RNAi agent.

In one embodiment, the RNAi agent is ligated to one or more diagnosticcompound, reporter group, cross-linking agent, nuclease-resistanceconferring moiety, natural or unusual nucleobase, lipophilic molecule,cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin,terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 60% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 70% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 80% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression ofthe Beta-ENaC gene by at least about 90% at a concentration of 10 nM inH441 cells in vitro.

In one embodiment, the RNAi has an EC50 of no more than about 0.1 nM.

In one embodiment, the RNAi has an EC50 of no more than about 0.01 nM.

In one embodiment, the RNAi has an EC50 of no more than about 0.001 nM.

A Method of Treatment Using a RNAi Agent Described Herein

In one particular specific embodiment, the present disclosure relates toa method of treating a Beta-ENaC-related disease in an individual,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising a RNAi agent comprising atleast an antisense strand, wherein the antisense strand comprises atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotidesfrom the antisense strand of a RNAi agent to Beta-ENaC selected from thespecific duplexes provided herein and as listed, e.g., in Table 1. Inanother embodiment, the present disclosure relates to such method,wherein the composition comprising a RNAi agent further comprises asense strand, wherein the sense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the sense strandof a RNAi agent to Beta-ENaC selected from the specific duplexesprovided herein and as listed, e.g., in Table 1.

Various particular specific embodiments of this embodiment are describedbelow.

In one embodiment, the Beta-ENaC-related disease is cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and/or obesity-associated hypertension.

In one embodiment, the Beta-ENaC-related disease is cystic fibrosis.

In one embodiment, the method further comprises the administration of anadditional treatment. In one embodiment, the additional treatment is atherapeutically effective amount of a composition.

In one embodiment, the additional treatment is a method (or procedure).

In one embodiment, the additional treatment and the RNAi agent can beadministered in any order, or can be administered simultaneously.

In one embodiment, the method further comprises the step ofadministering an additional treatment for cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and/or obesity-associated hypertension.

In one embodiment, the method further comprises the step ofadministering an additional treatment or therapy selected from the listof an additional antagonist to ENaC, a potassium-sparing diuretic,amiloride, triamterene, regulation of dietary salt intake, antibiotics,DNase therapy, albutrol, N-acetylcysteine, breathing therapy, percussivetherapy, and aerobic exercise.

In one embodiment, the composition comprises a second RNAi agent toBeta-ENaC. In various embodiments, the second RNAi agent is physicallyseparate from the first, or the two are physically connected (e.g.,covalently linked or otherwise conjugated).

In one embodiment, the method further comprises the step ofadministering an additional RNAi agent which comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense strand of a RNAi agent to Beta-ENaC selected from the specificduplexes provided herein and as listed, e.g., in Table 1.

A Method of Inhibiting the Expression of Beta-ENaC, Using a RNAi AgentDescribed Herein

In one particular specific embodiment, the present disclosure relates toa method of inhibiting the expression of the Beta-ENaC gene in anindividual, comprising the step of administering to the individual atherapeutically effective amount of a composition comprising a RNAiagent of the present disclosure. In one embodiment, the RNAi agentcomprises at least an anti-sense strand, and/or comprises a sense and ananti-sense strand, wherein the anti-sense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theanti-sense strand of a RNAi agent to Beta-ENaC selected from thosespecific duplex provided herein and as listed, e.g., in Table 1.

Various embodiments of this aspect of the invention are described below.

In one embodiment, the individual is afflicted with or susceptible to aBeta-ENaC-related disease.

In one embodiment, the Beta-ENaC-related disease is cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and/or obesity-associated hypertension.

In one embodiment, the Beta-ENaC-related disease is cystic fibrosis.

In one embodiment, the method further comprises the administration of anadditional treatment. In one embodiment, the additional treatment is atherapeutically effective amount of a composition.

In one embodiment, the additional treatment is a method (or procedure).

In one embodiment, the additional treatment and the RNAi agent can beadministered in any order or can be administered simultaneously.

In one embodiment, the method further comprises the step ofadministering an additional treatment for cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and/or obesity-associated hypertension.

In one embodiment, the method further comprises the step ofadministering an additional treatment or therapy selected from the listof an additional antagonist to ENaC, a potassium-sparing diuretic,amiloride, triamterene, regulation of dietary salt intake, antibiotics,DNase therapy, albutrol, N-acetylcysteine, breathing therapy, percussivetherapy, and aerobic exercise.

In one embodiment, the composition comprises a second RNAi agent toBeta-ENaC. In various embodiments, the second RNAi agent is physicallyseparate from the first, or the two are physically connected (e.g.,covalently linked or otherwise conjugated).

In one embodiment, the method further comprises the step ofadministering an additional RNAi agent which comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense strand of a RNAi agent to Beta-ENaC selected from the specificduplexes provided herein and as listed, e.g., in Table 1.

Pharmaceutical Formulations of a RNAi Agent to Beta-ENaC

In one particular specific embodiment, the present disclosure relates toa composition comprising a RNAi agent of the present disclosure. In oneembodiment, the RNAi agent comprises at least an anti-sense strand,and/or comprises a sense and an anti-sense strand, wherein theanti-sense strand comprises at least 15 contiguous nucleotides differingby 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agentto Beta-ENaC selected from those specific duplex provided herein and aslisted, e.g., in Table 1, wherein the composition is in apharmaceutically effective formulation.

In one embodiment, the present disclosure pertains to the use of a RNAiagent in the manufacture of a medicament for treatment of aBeta-ENaC-related disease, wherein the RNAi agent comprises a sensestrand and an antisense strand, wherein the antisense strand comprisesat least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the antisense strand of a RNAi agent to Beta-ENaCselected from those specific duplex provided herein and as listed, e.g.,in Table 1.

ENaC

By “ENaC” is meant the epithelial sodium channel, a membrane proteinmade of three different but homologous subunits (Alpha, Beta and Gamma).

ENaC is present in the apical membrane of epithelial cells of the distalnephron (cortical and medullary collecting tubule) and distal colon andin the airways and in the excretory ducts of several glands. ENaC isalso expressed in placenta, brain and urinary bladder. It provides acontrolled entry pathway for Na⁺ from the lumen of these organs into theepithelial cells, and, together with the Na⁺/K⁺-ATPase located in thebasolateral membrane of the same cells, and is responsible for theactive, vectorial transport of Na⁺ from the external medium through theepithelial cells into the extracellular fluid and toward the blood. ENaCis located on the apical membrane facing the lumen, and allows movementof sodium from the lumen into the epithelial cell. The sodium reabsorbedvia ENaC is then extruded from the epithelial cell back into thebloodstream by the Na⁺/K⁺-ATPase. The reabsorption of sodium by the ENaCis accompanied by an osmotic uptake of water to maintain a constantextracellular Na⁺ concentration. This changes blood volume andconsequently affects blood pressure. Thus, ENaC plays an important rolein electrolyte homeostasis and the control of blood volume and bloodpressure. See, e.g., Saxena et al. 1998 Biochem. Biophys. Res. Comm.252: 208-213.

ENaC has different functional roles in various organs in which it isexpressed. In the kidney (collecting tubule), the modulated reabsorptionof Na⁺ through ENaC provides the primary mechanism of the regulation ofurinary Na⁺ excretion and thus allows the fine control of the wholeorganism Na⁺ balance under the hormonal control of aldosterone. By itsdepolarizing effect on the apical membrane potential, the Na⁺ channelalso provides the driving force for tubular K⁺ secretion.

Specific inhibitors of ENaC promote urinary Na⁺ excretion and inhibit K⁺secretion; these drugs (including amiloride and triamterene), aretherefore used as K⁺-sparing diuretics. ENaC has a similar functionalrole in the distal colon, preventing excessive Na⁺ loss in the stools.In airways, an important role is the reabsorption of the fluid thatfills the airways at birth, promoting the shift from fluid secretion(before birth) to fluid reabsorption (postnatal).

With the cystic fibrosis transmembrane conductance regulator, it alsoparticipates in the delicate regulation of the fluid balance in theairways that maintains a thin mucosal fluid film necessary for mucusclearance. In the excretory ducts of the salivary and sweat glands, theactivity of ENaC tends to decrease the luminal Na⁺ concentration,allowing the excretion of a less salty saliva and preventing major lossof Na⁺ in the sweat fluid. See, for example, Hummler et al. 1999 Am. J.Physiol. Gastrointest. Liver Physiol. 276: 567-571 and references citedtherein.

Alterations and mutations in the sequence and/or expression of ENaC canlead to over-expression or hyper-activity of ENaC. Providing RNAi agentsof this disclosure restores balance to the modulated reabsorption of Na⁺by reducing the level of the Beta-ENaC.

Beta-ENaC

By “Beta-ENaC” is meant the gene or protein amiloride-sensitive sodiumchannel subunit beta (or any nucleic acid encoding this protein), alsovariously designated: sodium channel, nonvoltage-gated 1, beta; SCNN1B;bENaC; ENaCb; ENaC-beta; SCNEB, or β-ENaC. Additional identifiersinclude: OMIM: 600760; MGI: 104696; HomoloGene: 284; and GeneCards:SCNN1B. Additional information can be found: Human: Entrez 6338; EnsemblENSG00000168447; UniProt P51168; RefSeq (mRNA) NM_(—)000336; RefSeq(protein) NP_(—)000327; Location (UCSC) Chr 16: 23.22-23.3 Mb. Mouse:Entrez 20277; Ensembl ENSMUSG00000030873; UniProt Q3TP51; RefSeq (mRNA)NM_(—)011325 RefSeq (protein) NP_(—)035455; Location (UCSC) Chr 7:121.66-121.71 Mb.

The amino acid sequence of human Beta-ENaC is provided in Saxena et al.1998 Biochem. Biophys. Res. Comm. 252: 208-213.

The functional domains of Beta-ENaC have been delineated. The proteinhas an intracellular N-terminal domain [amino acids (“aa”) 1 to 50], afirst transmembrane domain (aa 51 to 71), an extracellular loop (aa 72to 533), a second transmembrane domain (aa 534 to 553), and a C-terminalintracellular domain (aa 554 to 640).

The C-terminal intracellular domain contains two regions whereinmutations relate to Liddle's syndrome and other diseases: in the regionfrom amino acid 564 to 595 and the “PY” motif [with the amino acidconsensus sequence PPXY at aa (amino acids) 615 to 618]. See, e.g.,Saxena et al. 1998.

The Beta-ENaC RNAi agent of the present disclosure can interact with aportion of the mRNA corresponding to a specific functional domain ordomains of Beta-ENaC. In various embodiments, the RNAi agents hereinspecifically bind to Beta-ENaC mRNA, in a sequence corresponding to afunctional domain, e.g., in the N-terminal intracellular domain, in thefirst transmembrane domain, in the extracellular loop, in the secondtransmembrane domain, or in the C-terminal intracellular domain, or,more specifically, in the region from amino acid 564 to 595, or in thePY motif (amino acids 615 to 618).

In various embodiments, the RNAi agents of the present disclosure bindto the 5′ or 3′ UTR (i.e., untranslated region(s)).

In various embodiments, the RNAi agents of the present disclosure bindto Beta-ENaC mRNA, but not in a sequence corresponding to a particularfunctional domain, e.g., not in the N-terminal intracellular domain, notin the first transmembrane domain, not in the extracellular loop, not inthe second transmembrane domain, or not in the C-terminal intracellulardomain, or, more specifically, not in the region from amino acid 564 to595, or not in the PY motif (amino acids 615 to 618).

In embodiments herein, binding of a RNAi agent to a particular region ofthe Beta-ENaC mRNA leads to reduced expression, level and/or activity ofBeta-ENaC.

The efficacy of a RNAi agent in reducing the level of Beta-ENaC can bemeasured directly, e.g., by measuring the levels of Beta-ENaC mRNAabundance or levels of the protein itself. Alternatively, the efficacyof the RNAi can be measured indirectly by measuring the level of any oneor more of the known activities of Beta-ENaC or by measuring changes inthe activities of pathway components downstream of Beta-ENaC.

The protein's chief activity is to form, along with Alpha-ENaC andGamma-ENaC, and, possibly at times, Delta-ENaC, the sodium channel ENaC.Beta-ENaC, Gamma-ENaC and Delta-ENaC may also form a particular type ofchannel found in the pancreas, testes and ovaries. Beta-ENaC has alsobeen shown to interact with WWP2 and NEDD4. See., e.g., McDonald et al.(2002). Am. J. Physiol. Renal Physiol. 283 (3): F431-6; Harvey et al.2001. J. Biol. Chem. 276 (11): 8597-601; Farr et al. (2000). Biochem. J.345 Pt 3: 503-9. The activity of Beta-ENaC can be measured, for example,by its ability to bind and form functional units with these otherbiological components. The efficacy of a RNAi agent can also be measuredindirectly by measuring the amount of surface liquid on mucus membranes,and via histological studies of tissues expressing Beta-ENaC.

Beta-ENaC Sequences in Various Species

A RNAi agent specific to Beta-ENaC can be designed such that thesequence thereof completely matches that of the mRNA corresponding tothe human Beta-ENaC gene and the homologous gene from a test animal.Thus, the exact same RNAi agent can be used in both test animals (e.g.,rat, mouse, cynomolgus monkey, etc.) and humans. The sequences for thevarious ENaC genes have been determined in many species, includinghumans, mouse, rat, bovine and chicken, as described in, inter alia,Garty et al. 1997 Physiol. Rev. 77: 359-396; and Ahn et al. 1999 Am. J.Physiol. 277:F121-F129.

The Beta-ENaC sequence in cynomolgus monkey (Macaca fascicularis, or“cyno”) has been determined.

The alignment of the cyno Beta-ENaC mRNA (SEQ ID NO: 221) and human Beta- ENaC mRNA (SEQ ID NO: 222) sequences is shown below. Cyno Beta-ENaC-------------------------------------------------- Human Beta-ENaCGTGCTTCCCCGCCCCTGAACCTGCTCCCTCCCAGTCGGTCTCGCCGCGCT   50 Cyno Beta-ENaC-----------------------------------GGTACCCAGCTTGCT   15 Human Beta-ENaCCGCCGGGTGTCCCAGTGTCACCAACACTCGGCCGCCGCCGCCAGCTTGGC  100                                    *   ******** Cyno Beta-ENaCTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGCAGATCAAT   65 Human Beta-ENaCGCGCACCGCCGCCTCCGCCACCGCCGACAGCGCGCATCCTCCGTGTCCCC  150          **    **        *  ** *   *    *   ** Cyno Beta-ENaCTCCCCGGGGATCCGA-ATTCGCCACCATGCACGTGAAGAAGTACCTGCTG  114 Human Beta-ENaCGCTCCGCCGCCCGAGCAGGTGCCACTATGCACGTGAAGAAGTACCTGCTG  200 * ***  *  *    *   ***** ************************ Cyno Beta-ENaCAAGTGCCTGCACCGGCTGCAGAAGGGCCCCGGCTACACGTACAAGGAGCT  164 Human Beta-ENaCAAGGGCCTGCATCGGCTGCAGAAGGGCCCCGGCTACACGTACAAGGAGCT  250*** ******* ************************************** Cyno Beta-ENaCGCTGGTGTGGTACTGCGATAACACCAACACCCACGGCCCCAAGCGTATCA  214 Human Beta-ENaCGCTGGTGTGGTACTGCGACAACACCAACACCCACGGCCCCAAGCGCATCA  300****************** ************************** **** Cyno Beta-ENaCTCTGCGAGGGGCCCAAGAAGAAAGCCGTGTGGTTCCTGCTCACCCTGCTC  264 Human Beta-ENaCTCTGTGAGGGGCCCAAGAAGAAAGCCATGTGGTTCCTGCTCACCCTGCTC  350**** ********************* *********************** Cyno Beta-ENaCTTCACTGCTCTCGTCTGCTGGCAGTGGGGCATCTTCATCAGGACCTACTT  314 Human Beta-ENaCTTCGCCGCCCTCGTCTGCTGGCAGTGGGGCATCTTCATCAGGACCTACTT  400*** * ** ***************************************** Cyno Beta-ENaCGAGCTGGGAGGTCAGCGTCTCCCTCTCCGTAGGCTTCAAGACCATGGACT  364 Human Beta-ENaCGAGCTGGGAGGTCAGCGTCTCCCTCTCCGTAGGCTTCAAGACCATGGACT  450************************************************** Cyno Beta-ENaCTCCCCGCCGTCACCATCTGCAATGCTAGCCCCTTCAAGTATTCCAAAGTC  414 Human Beta-ENaCTCCCTGCCGTCACCATCTGCAATGCTAGCCCCTTCAAGTATTCCAAAATC  500**** ****************************************** ** Cyno Beta-ENaCAAGCATTTGCTGAAGGACCTGGATGAGCTGATGGAAGCTGTCCTGGAGAG  464 Human Beta-ENaCAAGCATTTGCTGAAGGACCTGGATGAGCTGATGGAAGCTGTCCTGGAGAG  550************************************************** Cyno Beta-ENaCAATCCTGGCTCCTGAGCTAAGCCATGCCAATGCCACCAGGACCCTGAACT  514 Human Beta-ENaCAATCCTGGCTCCTGAGCTAAGCCATGCCAATGCCACCAGGAACCTGAACT  600***************************************** ******** Cyno Beta-ENaCCTTCCATCTGGAACCACACACCACTGGTCCTTATTGATGAACGGAACCCC  564 Human Beta-ENaCTCTCCATCTGGAACCACACACCCCTGGTCCTTATTGATGAACGGAACCCC  650  ******************** *************************** Cyno Beta-ENaCCACCACCCCATGGTCCTCGATCTCTTTGGAGATAACCACAATGGCTTAAC  614 Human Beta-ENaCCACCACCCCATGGTCCTTGATCTCTTTGGAGACAACCACAATGGCTTAAC  700***************** ************** ***************** Cyno Beta-ENaCAAACAGCTCAGCATCAGAAAAGATCTGTAATGCCCATGGGTGCAAAATGG  664 Human Beta-ENaCAAGCAGCTCAGCATCAGAAAAGATCTGTAATGCCCACGGGTGCAAAATGG  750** ********************************* ************* Cyno Beta-ENaCCCATGAGACTATGTAGCCTCAACGGGACCCAGTGCACCTTCCGGAACTTC  714 Human Beta-ENaCCCATGAGACTATGTAGCCTCAACAGGACCCAGTGTACCTTCCGGAACTTC  800*********************** ********** *************** Cyno Beta-ENaCACCAGCGCTACCCAGGCAGTGACAGAGTGGTACAGCCTGCAGGCCACCAA  764 Human Beta-ENaCACCAGTGCTACCCAGGCATTGACAGAGTGGTACATCCTGCAGGCCACCAA  850***** ************ *************** *************** Cyno Beta-ENaCCATCTTTGCGCAGGTGCCGCAGCAGGAGCTGGTGGAGATGAGCTACCCCG  814 Human Beta-ENaCCATCTTTGCACAGGTGCCACAGCAGGAGCTAGTAGAGATGAGCTACCCCG  900********* ******** *********** ** **************** Cyno Beta-ENaCGCGAGCAGATGATCCTGGCCTGCCTGTTTGGAGCTGAGCCCTGCAACTAC  864 Human Beta-ENaCGCGAGCAGATGATCCTGGCCTGCCTATTCGGAGCTGAGCCCTGCAACTAC  950************************* ** ********************* Cyno Beta-ENaCCGGAACTTCACGTCCATCTTCTACCCTCACTATGGCAACTGTTACATCTT  914 Human Beta-ENaCCGGAACTTCACGTCCATCTTCTACCCTCACTATGGCAACTGTTACATCTT 1000************************************************** Cyno Beta-ENaCCAACTGGGGCATGACAGAGAAGGCACTTCCTTCGGCCAACCCTGGACCTG  964 Human Beta-ENaCCAACTGGGGCATGACAGAGAAGGCACTTCCTTCGGCCAACCCTGGAACTG 1050********************************************** *** Cyno Beta-ENaCAATTTGGCCTGAAGTTGATCCTGGACATAGGCCAGGAAGACTACGTCCCC 1014 Human Beta-ENaCAATTCGGCCTGAAGTTGATCCTGGACATAGGCCAGGAAGACTACGTCCCC 1100**** ********************************************* Cyno Beta-ENaCTTCCTCGCGTCCACGGCTGGGGTCAGGCTGATGCTTCACGAGCAGAGGTC 1064 Human Beta-ENaCTTCCTTGCGTCCACGGCCGGGGTCAGGCTGATGCTTCACGAGCAGAGGTC 1150***** *********** ******************************** Cyno Beta-ENaCATACCCCTTCATCAGAGACGAGGGCATCTATGCCATGTCGGGGACAGAGA 1114 Human Beta-ENaCATACCCCTTCATCAGAGATGAGGGCATCTACGCCATGTCGGGGACAGAGA 1200****************** *********** ******************* Cyno Beta-ENaCCGTCCATCGGGGTACTCGTGGACAAGCTTCAGCGCATGGGGGAGCCCTAC 1164 Human Beta-ENaCCGTCCATCGGGGTACTCGTGGACAAGCTTCAGCGCATGGGGGAGCCCTAC 1250************************************************** Cyno Beta-ENaCAGCCCGTGCACCGTGAATGGCTCCGAGGTCCCCGTCCAAAACTTCTACAG 1214 Human Beta-ENaCAGCCCGTGCACCGTGAATGGTTCTGAGGTCCCCGTCCAAAACTTCTACAG 1300******************** ** ************************** Cyno Beta-ENaCTGACTACAACACGACCTACTCCATCCAGGCCTGTCTTCGCTCCTGCTTCC 1264 Human Beta-ENaCTGACTACAACACGACCTACTCCATCCAGGCCTGTCTTCGCTCCTGCTTCC 1350************************************************** Cyno Beta-ENaCAAGACCACATGATCCGTAGCTGCAAGTGTGGGCACTACCTCTACCCACTG 1314 Human Beta-ENaCAAGACCACATGATCCGTAACTGCAACTGTGGCCACTACCTGTACCCACTG 1400****************** ****** ***** ******** ********* Cyno Beta-ENaCCCCCGTGGGGAGAAATACTGCAACAACCGGGACTTCCCAGACTGGGCCCA 1364 Human Beta-ENaCCCCCGTGGGGAGAAATACTGCAACAACCGGGACTTCCCAGACTGGGCCCA 1450************************************************** Cyno Beta-ENaCTTGCTACTCAGATCTGCAGATGAGCGTGGCGCAGAGAGAGACCTGCATTG 1414 Human Beta-ENaCTTGCTACTCAGATCTACAGATGAGCGTGGCGCAGAGAGAGACCTGCATTG 1500*************** ********************************** Cyno Beta-ENaCGCATGTGCAAGGAATCCTGCAATGACACCCAGTACAAGATGACTATCTCC 1464 Human Beta-ENaCGCATGTGCAAGGAGTCCTGCAATGACACCCAGTACAAGATGACCATCTCC 1550************* ***************************** ****** Cyno Beta-ENaCATGGCTGACTGGCCTTCTGAGGCCTCTGAGGACTGGATTTTCCACGTCTT 1514 Human Beta-ENaCATGGCTGACTGGCCTTCTGAGGCCTCCGAGGACTGGATTTTCCACGTCTT 1600************************** *********************** Cyno Beta-ENaCGTCTCAGGAGCGGGACCAAAGCACCAATATCACCCTGAGCAGGAAGGGAA 1564 Human Beta-ENaCGTCTCAGGAGCGGGACCAAAGCACCAATATCACCCTGAGCAGGAAGGGAA 1650************************************************** Cyno Beta-ENaCTTGTCAAGCTCAACATCTACTTCCAAGAATTTAACTATCGCACCATTGAA 1614 Human Beta-ENaCTTGTCAAGCTCAACATCTACTTCCAAGAATTTAACTATCGCACCATTGAA 1700************************************************** Cyno Beta-ENaCGAATCAGCAGCCAATAACCTCGTCTGGCTGCTCTCAAATCTGGGTGGCCA 1664 Human Beta-ENaCGAATCAGCAGCCAATAACATCGTCTGGCTGCTCTCGAATCTGGGTGGCCA 1750****************** **************** ************** Cyno Beta-ENaCGTTTGGCTTCTGGATGGGGGGCTCTGTGCTGTGCCTCATCGAGTTTGGGG 1714 Human Beta-ENaCGTTTGGCTTCTGGATGGGGGGCTCTGTGCTGTGCCTCATCGAGTTTGGGG 1800************************************************** Cyno Beta-ENaCAGATCATCATCGACTTTGTGTGGATCACCATCATCAAGCTGGTGGCCTTG 1764 Human Beta-ENaCAGATCATCATCGACTTTGTGTGGATCACCATCATCAAGCTGGTGGCCTTG 1850************************************************** Cyno Beta-ENaCGCCAAGAGCCTCCGGCAGCGGCGAGCCCAAGCCAGCTACTCCGGCCCACC 1814 Human Beta-ENaCGCCAAGAGCCTACGGCAGCGGCGAGCCCAAGCCAGCTACGCTGGCCCACC 1900*********** *************************** * ******** Cyno Beta-ENaCGCCCACGGTGGCTGAGCTGGTGGAGGCCCACACCAACTTCGGCTACCAGC 1864 Human Beta-ENaCGCCCACCGTGGCCGAGCTGGTGGAGGCCCACACCAACTTTGGCTTCCAGC 1950****** ***** ************************** **** ***** Cyno Beta-ENaCCTGACACGGCCCCCCGCAGCCCCAACACCGGGCCCTACCCCAGTGAGCAG 1914 Human Beta-ENaCCTGACACGGCCCCCCGCAGCCCCAACACTGGGCCCTACCCCAGTGAGCAG 2000**************************** ********************* Cyno Beta-ENaCGCCCTGCCCATCCCGGGCACCCCGCCCCCCAACTATGACTCCCTGCGTCT 1964 Human Beta-ENaCGCCCTGCCCATCCCAGGCACCCCGCCCCCCAACTATGACTCCCTGCGTCT 2050************** *********************************** Cyno Beta-ENaCGCAGCCACTGGACGTCATCGAGTCTGACAGTGAGGGTGATGCCATCTAA- 2013 Human Beta-ENaCGCAGCCGCTGGACGTCATCGAGTCTGACAGTGAGGGTGATGCCATCTAAC 2100****** ****************************************** Cyno Beta-ENaC---GCGGCCGCCTAG---AAATAGCTTGATCTGGTTA--CCACTAAACCA 2055 Human Beta-ENaCCCTGCCCCTGCCCACCCCGGGCGGCTGAAACTCACTGAGCAGCCAAGACT 2150   **  * *** *         ***  * **   *   *  * **  * Cyno Beta-ENaCGC--CTCAAGAACAC-CCGAATGGAGTCTCT----AAGCTACATAATACC 2098 Human Beta-ENaCGTTGCCCGAGGCCTCACTGTATGGTGCCCTCTCCAAAGGGTCGGGAGGGT 2200*   * * **  * * * * **** * *       ***   *   * Cyno Beta-ENaCAACTTACACTTTACAAAATGTTGTCCCCCAA-AATGTAGCCATTCGTATC 2147 Human Beta-ENaCAGCTCTCCAGGCCAGAGCTTGTGTCCTTCAACAGAGAGGCCAGCGGCAAC 2250* **  *        *  *  *****  *** *  *  ****   * * * Cyno Beta-ENaCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTAAAAAAAAAAAAAAAA 2197 Human Beta-ENaCTGGTCCGTTACTGGCCAAGGGCTCTGTAGAATCACGGTGCTGGTACAGGA 2300** ***          ** *  ***  * * **           * *  * Cyno Beta-ENaCAAAAAAAAAAAAAAAAAACCCCCCCC--CCCCCCCCCCTGCAGAGATCTG 2245 Human Beta-ENaCTGCAGGAATAAATTGTATCTTCACCTGGTTCCTACCCTCGTCCCTACCTG 2350   *  ** ***    * *  * **     **  ***  *     * *** Cyno Beta-ENaCCTAGCTTGAGTATTCTATAGAGTCACCTAAATACT--------------- 2280 Human Beta-ENaCTCCTGATCCTGGTCCTGAAGACCCCTCGGAACACCCTCTCCTGGTGGCAG 2400      *     * **  ***  *  *  ** ** Cyno Beta-ENaC-------------------------------------------------- Human Beta-ENaCGCCACTTCCCTCCCAGTGCCAGTCTCCATCCACCCCAGAGAGGAACAGGC 2450 Cyno Beta-ENaC-------------------------------------------------- Human Beta-ENaCGGGTGGGCCATGTGGTTTTCTCCTTCCTGGCCTTGGCTGGCCTCTGGGGC 2500 Cyno Beta-ENaC-------------------------------------------------- Human Beta-ENaCAGGGGTGGTGGAGAGATGGAAGGGCATCAGGTGTAGGGACCCTGCCAAGT 2550 Cyno Beta-ENaC----------------------------------------------- Human Beta-ENaCGGCACCTGATTTACTCTAGAAAATAAAAGTAGAAAATACTGAGTCCA 2597 Cyno Beta-ENaC(SEQ ID NO: 221) Human Beta-ENaC (SEQ ID NO: 222)

The start (ATG) and stop (TAA) codons of the cyno and human sequencesare underlined. Nucleotides matching between the human and cynosequences are marked with an asterisk (*).

In one embodiment, the Beta-ENaC RNAi agent of the present disclosurecomprises a sequence which is identical in the human, rat and cynoBeta-ENaC mRNAs. This sequence identity facilitates animal testing priorto human testing. In another embodiment, the Beta-ENaC RNAi agentcomprises a sequence which is identical in the human, mouse and cynoBeta-ENaC mRNAs.

Additional Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the Beta-ENaC RNAi agent comprises a sequence whichdoes not match that of any other mRNA or gene. In one embodiment, theBeta-ENaC RNAi agent comprises a sequence which differs from all otherknown non-Beta-ENaC mRNAs or genes by at least 0, 1, 2 or 3 nucleotides.

In one embodiment, the Beta-ENaC RNAi agent of the present disclosure isadministered to a patient in need thereof (e.g., a patient sufferingfrom cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle'ssyndrome, hypertension, alkalosis, hypokalemia, and obesity-associatedhypertension).

The patient can also be administered more than one RNAi agent specificto Beta-ENaC. In one embodiment, the Beta-ENaC RNAi agent(s) of thepresent disclosure can optionally be administered along with one or moreadditional pharmaceutical agent appropriate for that disease. In oneembodiment, the Beta-ENaC RNAi agent(s) of the present disclosure can beoptionally administered along with any other appropriate additionaltreatment, wherein the additional treatment can be a composition or amethod.

In the case of cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1),Liddle's syndrome, hypertension, alkalosis, hypokalemia, and/orobesity-associated hypertension, the RNAi agent(s) and additionaldisease treatment(s) can be administered in any order, simultaneously orsequentially, or in one or multiple doses over time.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

RNAi Agent

In one embodiment, the present disclosure pertains to a Beta-ENaC RNAiagent or other composition comprising at least an antisense nucleic acidsequence complementary to a Beta-ENaC nucleic acid (or portion thereof),or pertains to a recombinant expression vector encoding the siRNA orcomposition comprising the antisense nucleic acid that can function asan RNAi as defined below. As used herein, an “antisense” nucleic acidcomprises a nucleotide sequence complementary to a “sense” nucleic acidencoding the Beta-ENaC protein (e.g., complementary to the coding strandof a double-stranded DNA, complementary to an mRNA or complementary tothe coding strand of a Beta-ENaC gene or nucleic acid).

As used herein, the term “RNAi agent to Beta-ENaC,” “RNAi agent specificto Beta-ENaC,” “iRNA agent to Beta-ENaC,” “siRNA to Beta-ENaC”,“Beta-ENaC siRNA” and the like refer to a siRNA (short inhibitory RNA),shRNA (short or small hairpin RNA), iRNA (interference RNA) agent, RNAi(RNA interference) agent, dsRNA (double-stranded RNA), microRNA, and thelike, and refer to a composition which specifically targets, is specificto, and/or binds to a Beta-ENaC mRNA. As used herein, the term“antisense nucleic acid” or “composition comprising an anti-sensenucleic acid” and the like is broadly meant to encompass any compositioncomprising at least one nucleic acid strand which is anti-sense to itstarget; this includes, but is not limited to, any siRNA, shRNA, iRHA,dsRNA, microRNA, antisense oligonucleotide, and any other compositioncomprising an anti-sense nucleic acid. As used herein, the terms “iRNA”and “RNAi” refers to an agent that contains RNA (or a derivativethereof), and which mediates the targeted cleavage of another RNAtranscript via an RNA-induced silencing complex (RISC) pathway. In oneembodiment, the RNAi agent is an oligonucleotide composition thatactivates the RISC complex/pathway. In another embodiment, the RNAiagent comprises an antisense strand sequence (antisenseoligonucleotide).

The RNAi agent(s) of the present disclosure target (e.g., bind to,anneal to, etc.) the Beta-ENaC mRNA. The use of the RNAi agent specificto Beta-ENaC results in a decrease of Beta-ENaC activity, level and/orexpression, e.g., a “knock-down” or “knock-out” of the target gene ortarget sequence. Particularly in one embodiment, in the case of adisease state characterized by over-expression or hyper-activity ofBeta-ENaC, administration of a RNAi agent to Beta-ENaC knocks down theBeta-ENaC target enough to restore a normal level of Beta-ENaC activityand/or a normal level of Na⁺ reabsorption.

In one embodiment, the RNAi comprises a single strand (such as an shRNA,as described herein).

In various embodiments, one or both strands are nicked.

In one embodiment, a single-stranded RNAi agent oligonucleotide orpolynucleotide can comprise the sense and/or antisense strand. See,e.g., Sioud 2005 J. Mol. Biol. 348:1079-1090, and references citedtherein. Thus the present disclosure encompasses RNAi agents with asingle strand comprising either the sense or antisense strand of a RNAiagent described herein.

siRNAs that are particularly useful for this disclosure include thosewhich can bind specifically to a region of the Beta-ENaC mRNA, and haveone or more of the following qualities: binding in the coding segment ofBeta-ENaC; binding at or near the junction of the 5′ untranslated regionand the start of the coding segment; binding at or near thetranslational start site of the mRNA; binding at, across or nearjunctions of exons and introns; little or no binding to the mRNAs ortranscripts of other genes (little or no “off-target effects”); bindingto the Beta-ENaC mRNA in or near a region or regions that is notdouble-stranded or a stem region, e.g., in a loop or single-strandedportion; eliciting little or no immunogenicity; binding in a segment ofthe Beta-ENaC mRNA sequence which is conserved among various animalspecies (including human, mouse, rat, cyno, etc.), as the presence of aconserved sequence facilitates testing using various laboratory animals;binding to double-stranded region(s) of the mRNA; binding to an AT-richregion (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or60% AT-rich); and/or lacking particular sequences known or suspected todecrease siRNA activity, e.g., the presence of a GG sequence at the 5′end, which may decrease separation of the double-stranded portion of thesiRNA. In one embodiment, the RNAi agent specific to Beta-ENaC can be adouble-stranded RNA having any one or more of these qualities.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aRNAi agent comprising a first and a second strand; e.g., a compositionthat includes an RNA molecule or complex of molecules having ahybridized duplex region (i.e., a region where the nucleotide bases fromthe first strand and the second strand are paired) that comprises twoanti-parallel and substantially complementary nucleic acid strands,which will be referred to as having “sense” and “antisense” orientationswith respect to a target RNA. The antisense strand, with respect to themRNA target, is also called the “guide” strand, and the sense strand isalso called the “passenger” strand. The passenger strand can include atleast one or more of the following: one or more extra nucleotides (e.g.,a bulge or 1 nt loop) compared to the other strand, a nick, a gap, etc.,compared to the other strand. In various embodiments, the RNAi agentcomprises a first strand and a second strand. In various embodiments,the first strand is the sense strand and the second strand is theanti-sense strand. In other embodiments, the first strand is theanti-sense strand, and the second strand is the sense strand.

The duplex region can be of any length that permits specific degradationof a desired target RNA through a RISC pathway, but will typically rangefrom 9 to 36 base pairs (“bp”) in length, e.g., 15-30 bp in length.Considering a duplex between 9 and 36 bp, the duplex can be any lengthin this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bpand any sub-range therebetween, including, but not limited to 15-30 bp,15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp,15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp,19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 20basepairs, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp,21 bp, 22 bp, or 23 bp. The dsRNAs generated in the cell by processingwith Dicer and similar enzymes are generally in the range of about 19 toabout 22 bp in length. One strand of the duplex region of a dsRNAcomprises a sequence that is substantially complementary to a region ofa target RNA. The two strands forming the duplex structure can be from asingle RNA molecule having at least one self-complementary duplexregion, or can be formed from two or more separate RNA molecules thathybridize to form the duplex. Where the duplex region is formed from twoself-complementary regions of a single molecule, the molecule can have aduplex region separated by a single-stranded chain of nucleotides(herein referred to as a “hairpin loop”, e.g., such as found in an shRNAconstruct) between the 3′-end of one strand and the 5′-end of therespective other strand forming the duplex structure. The hairpin loopcan comprise at least one unpaired nucleotide; in some embodiments thehairpin loop can comprise at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 20, atleast 23 or more unpaired nucleotides. Where the two substantiallycomplementary strands of a dsRNA are comprised by separate RNAmolecules, those molecules need not, but can be covalently connected.Where the two strands are connected covalently by a hairpin loop, theconstruct is generally referred to herein and in the art as a “shRNA”.Where the two strands are connected covalently by means other than ahairpin loop, the connecting structure is referred to as a “linker.”

RNA Interference

RNA interference (RNAi) is a post-transcriptional, targetedgene-silencing technique that uses double-stranded RNA (dsRNA) todegrade messenger RNA (mRNA) containing the same sequence as the dsRNA.The process of RNAi occurs when ribonuclease III (Dicer) cleaves thelonger dsRNA into shorter fragments called siRNAs. siRNAs (smallinterfering RNAs) are typically about 21 to 23 nucleotides long andcomprise about 19 base pair duplexes. The smaller RNA segments thenmediate the degradation of the target mRNA. Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control. Hutvagner et al. 2001, Science, 293, 834. TheRNAi response also features an endonuclease complex, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded mRNA complementary to the antisense strand of thesiRNA. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex.

In one aspect, an RNA interference agent includes a single-stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double-strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling oneof the now unpaired siRNA strands to act as a “guide” strand to guidetarget recognition (Nykanen, et al., (2001) Cell 107:309). Upon bindingof the antisense guide strand to the appropriate target mRNA, one ormore endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in oneaspect the present disclosure relates to a single-stranded RNA thatpromotes the formation of a RISC complex to effect silencing of thetarget gene.

RNA interference has also been studied in a variety of systems. Work inDrosophila embryonic lysates (Elbashir et al. 2001 EMBO J. 20: 6877 andTuschl et al. International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity in a variety of systems, including especially mammals. Thesestudies have shown that 21-nucleotide siRNA duplexes are most activewhen containing 3′-terminal dinucleotide overhangs. Substitution of the3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H)was tolerated. In addition, a 5′-phosphate on the target-complementarystrand of a siRNA duplex is usually required for siRNA activity. Mostimportantly for therapeutic use, siRNA duplexes shorter than 50 bp or sodo not activate the interferon response in mammalian cells. See, e.g.,Tuschl et al., WO 01/752164.

The dsRNA molecules (RNAi agents) described herein are thus useful inRNA interference of Beta-ENaC.

Features of a RNAi Agent: Sense Strand, Antisense Strand and (Optional)Overhangs

In various embodiments, the RNAi agents comprise a first strand and asecond strand, e.g., a sense strand and an antisense strand and,optionally, one or both ends of the duplex containing unpairednucleotides referred to herein as overhangs.

The term “antisense strand” refers to the strand of a RNAi agent whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally, the most tolerated mismatches are in theterminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a RNAiagent that includes a region that is substantially complementary to aregion of the antisense strand as that term is defined herein.

The sequence of a gene may vary from individual to individual,especially at wobble positions within the coding segment, or in theuntranslated region; individuals may also differ from each other incoding sequence, resulting in additional differences in mRNA. Thesequence of the sense and antisense strands of the RNAi agent can thusbe designed to correspond to that of an individual patient, if and whereneeded. RNAi agents can also be modified in sequence to reduceimmunogenicity, binding to undesired mRNAs (e.g., “off-target effects”)or to increase stability in the blood. These sequence variants areindependent of chemical modification of the bases or 5′ or 3′ or otherend-caps of the RNAi agents.

The RNAi agents can also have overhangs of 0, 1, or 2 overhangs; in thecase of a 0 nt overhang, they are blunt-ended. A RNAi agent can have 0,1 or 2 blunt ends. In a “blunt-ended RNAi agent” both strands terminatein a base-pair; thus a blunt-ended molecule lacks either 3′ or 5′single-stranded nucleotide overhangs.

As used herein, the term “overhang” or “nucleotide overhang” refer to atleast one unpaired nucleotide that protrudes from the end of at leastone of the two strands of the duplex structure of a RNAi agent. Forexample, when a 3′-end of one strand of a dsRNA extends beyond the5′-end of the other strand, or vice versa, the unpaired nucleotide(s)form the overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. An overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) may be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′ end, 3′ end or both ends of either an antisense orsense strand of a dsRNA.

The RNAi agent can also optionally comprise a cap. The term “cap” andthe like include a chemical moiety attached to the end of adouble-stranded nucleotide duplex, but is used herein to exclude achemical moiety that is a nucleotide or nucleoside. A “3′ Cap” isattached at the 3′ end of a nucleotide or oligonucleotide. A “5′ Cap” isattached at the 5′ end of a nucleotide or oligonucleotide. In oneembodiment, 3′ end caps are as disclosed in, for example, WO 2005/021749and WO 2007/128477.

The present disclosure thus contemplates a RNAi agent specific toBeta-ENaC comprising an antisense strand (which may be contiguous orconnected via a linker or loop) in a RNAi agent. In a more specificembodiment, an RNAi agent comprises an antisense strand and a sensestrand which together comprise a double-stranded or complementaryregion. In one embodiment, it can also optionally comprise one or twooverhangs and/or one or two caps. The RNAi agent is used to induce RNAinterference of Beta-ENaC.

Target and Complementary Sequences

The RNAi agents of the present disclosure target (e.g., specificallybind to, anneal to, etc.) the mRNA encoding the gene Beta-ENaC. The useof the RNAi agent specific to Beta-ENaC results in a decrease ofBeta-ENaC activity, level and/or expression, e.g., a “knock-down” or“knock-out” of the target gene or target sequence. Particularly in oneembodiment, in the case of a disease state characterized byover-expression or hyper-activity of Beta-ENaC, administration of a RNAiagent to Beta-ENaC knocks down the Beta-ENaC gene enough to restore anormal level of Beta-ENaC activity and/or a normal level of Na⁺reabsorption.

As used herein, “target sequence” or “target gene” refer to a contiguousportion of the nucleotide sequence of an mRNA molecule formed during thetranscription of a gene, e.g., a Beta-ENaC gene, including mRNA that isa product of RNA processing of a primary transcription product. Thetarget portion of the sequence will be at least long enough to serve asa substrate for iRNA-directed cleavage at or near that portion. Forexample, the target sequence will generally be from 9-36 nucleotides(“nt”) in length, e.g., 15-30 nt in length, including all sub-rangestherebetween. As non-limiting examples, the target sequence can be from15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt,15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt,18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, or 21-22nt, 21 nt, 22 nt, or 23 nt. The sense and antisense strands of the RNAicomprise a sequence complementary to that of the target nucleic acid,Beta-ENaC.

As used herein, and unless otherwise indicated, the term “complementary”refers to the ability of an oligonucleotide or polynucleotide comprisinga first nucleotide sequence to hybridize and form a duplex structureunder certain conditions with an oligonucleotide or polynucleotidecomprising a second nucleotide sequence. Such conditions can, forexample, be stringent, e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA,50° C. or 70° C. for 12-16 hours followed by washing. Other conditions,such as physiologically relevant conditions as may be encountered insidean organism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may furthermore be used with respect to the basematching between the sense strand and the antisense strand of a dsRNA,or between the antisense strand of a RNAi agent and a target sequence,as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding Beta-ENaC). For example, apolynucleotide is complementary to at least a part of a Beta-ENaC mRNAif the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding Beta-ENaC.

Complementary sequences within a RNAi agent, e.g., within a dsRNA asdescribed herein, include base-paired oligonucleotides orpolynucleotides comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they may form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single-stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a duplex comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein. The term overhang describes an unpaired nucleotide atthe 3′ or 5′ end of a double-stranded nucleotide duplex, as describedabove. In one embodiment, the overhang is 0 to 4 nt long and is on the3′ end.

Thus, the RNAi agent of the present disclosure is complimentary orsubstantially complimentary to a target sequence in the target Beta-ENaCand is double-stranded, comprising a sense and an antisense strand(which can be contiguous, linked via a loop, or otherwise joined), wherethe double-stranded region an be 9 to 36 bp long (particularly forexample, 19-22 bp or 19-23 bp long), and can furthermore optionallycomprise a 3′ or 5′ overhang, and the RNAi agent can furthermorecomprise a 3′ cap. The RNAi agent mediates RNA interference,down-regulating or inhibiting the level, expression and/or activity ofBeta-ENaC, and/or establishing or re-establishing an approximatelynormal level of ENaC and/or Beta-ENaC activity, or other biologicalfunction related to ENaC.

RNAi Agents Lowering Beta-ENaC Level, Expression and/or Activity

RNAi agents for targeting Beta-ENaC include those which bind to aBeta-ENaC sequence provided herein and which work to reduce Beta-ENaCthrough a RNAi mechanism. Exemplary siRNAs to Beta-ENaC are provided,e.g., in Table 1.

The RNAi agents of the present disclosure silence, inhibit theexpression of, down-regulate the expression of, and/or suppress theexpression of the Beta-ENaC gene, such that an approximately normallevel of Beta-ENaC activity, expression and/or level and/or Na⁺reabsorption is achieved.

In addition, in various embodiments, depending on the disease conditionand biological context, it is acceptable to use the RNAi agents of thepresent disclosure to establish a level of Beta-ENaC expression,activity and/or level which is below the normal level, or above thenormal level.

Any method known in the art can be use to measure changes in Beta-ENaCactivity, level and/or expression induced by a Beta-ENaC siRNA.Measurements can be performed at multiple timepoints, prior to, duringand after administration of the siRNA, to determine the effect of thesiRNA.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to a Beta-ENaC gene, herein refer to the at least partialsuppression of the expression of a Beta-ENaC gene, as manifested by areduction of the amount of Beta-ENaC mRNA which may be isolated from ordetected in a first cell or group of cells in which a Beta-ENaC gene istranscribed and which has or have been treated such that the expressionof a Beta-ENaC gene is inhibited, as compared to a second cell or groupof cells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

$\begin{matrix}{{\frac{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} ) - ( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} )}{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} )} \cdot 100}\%} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Beta-ENaC geneexpression, e.g., the amount of protein encoded by a Beta-ENaC gene,alteration in lung fluid levels or mucus levels, etc. In principle,Beta-ENaC gene silencing may be determined in any cell expressingBeta-ENaC, either constitutively or by genomic engineering, and by anyappropriate assay. However, when a reference or control is needed inorder to determine whether a given RNAi agent inhibits the expression ofthe Beta-ENaC gene by a certain degree and therefore is encompassed bythe instant disclosure, the assays provided in the Examples below shallserve as such reference.

For example, in certain instances, expression of a Beta-ENaC gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% by administration of a RNAi agent featured in the presentdisclosure. In some embodiments, a Beta-ENaC gene is suppressed by atleast about 60%, 70%, or 80% by administration of a RNAi agent featuredin the present disclosure. In some embodiments, a Beta-ENaC gene issuppressed by at least about 85%, 90%, or 95% or more by administrationof a RNAi agent, as described herein.

The ability of a RNAi agent to suppress Beta-ENaC can be first tested invitro (e.g., using test cells such as H441).

RNAi agents which can suppress Beta-ENaC in vitro can then be tested forimmunostimulation using, for example, a PBMC (peripheral bloodmononuclear cell) assay. RNAi agents can also be tested in animal tests.Test and control animals include those which over-express orunder-express Beta-ENaC, as described in, for example, Hummer et al.2005 J. Am. Soc. Nephrol. 16: 3160-3166; Randrianarison et al. 2007 Am.J. Physiol. Lung Cell. Mol. Physiol. 294: 409-416; Cao et al. 2006 Am.J. Physiol. Renal Physiol., and references cited therein. RNAi agentswhich suppress or alter the level, activity and/or expression ofBeta-ENaC can be used in medicaments to treat various Beta-ENaC-relateddiseases.

By “lower” in the context of Beta-ENaC or a symptom of aBeta-ENaC-related disease is meant a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least20%, at least 30%, at least 40% or more. If, for a particular disease,or for an individual suffering from a particular disease, the levels orexpression of Beta-ENaC are elevated, treatment with a Beta-ENaC RNAiagent of the present disclosure can particularly reduce the level orexpression of Beta-ENaC to a level considered in the literature aswithin the range of normal for an individual without such disorder. Thelevel or expression of Beta-ENaC can be measured by evaluation of mRNA(e.g., via Northern blots or PCR), or protein (e.g., Western blots). Theeffect of a RNAi agent on Beta-ENaC expression can be determined bymeasuring Beta-ENaC gene transcription rates (e.g., via Northern blots;or reverse transcriptase polymerase chain reaction or real-timepolymerase chain reaction). RT-PCR has been used to show that mRNAlevels of Beta-ENaC are high in kidney, pancreas and prostate, andmedium in liver and spleen. Brauner-Osborne et al. 2001. Biochim.Biophys. Acta 1518: 237-248. Direct measurements can be made of levelsof Beta-ENaC (which is expressed by the cell surface), e.g. by Westernblots of tissues in which Beta-ENaC is expressed.

As used herein, “down-regulates” refers to any statistically significantdecrease in a biological activity and/or expression of Beta-ENaC,including full blocking of the activity (i.e., complete inhibition)and/or expression. For example, “down-regulation” can refer to adecrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% inBeta-ENaC level, activity and/or expression.

As used herein, the term “inhibit” or “inhibiting” Beta-ENaC refers toany statistically significant decrease in biological level, activityand/or expression of Beta-ENaC, including full blocking of the activityand/or expression. For example, “inhibition” can refer to a decrease ofat least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% in Beta-ENaClevel, activity and/or expression. As used herein, the term “inhibit”similarly refers to a significant decrease in level, activity and/orexpression, while referring to any other biological agent orcomposition.

By “level”, it is meant that the Beta-ENaC RNAi agent can alter thelevel of Beta-ENaC, e.g., the level of Beta-ENaC mRNA or the level ofBeta-ENaC protein, or the level of activity of Beta-ENaC.

Some diseases, such as cystic fibrosis, are characterized by excessiveENaC-mediated Na⁺ absorption. Particularly in one embodiment, in thecase of a disease characterized by over-expression and/or hyper-activityof Beta-ENaC, administration of a RNAi agent to Beta-ENaC reduces thelevel, expression and/or activity of Beta-ENaC. However, excessively lowlevels of Beta-ENaC can also lead to impairment of lung fluid clearanceand renal dysfunction. Randrianarison et al. 2007 Am. J. Physiol. LungCell. Mol. Physiol. 294: 409-416. Thus, in various embodiments,administration of a RNAi agent to Beta-ENaC particularly establishes orre-establishes a normal or approximately normal level of Beta-ENaCactivity, expression and/or level.

By “normal” or “approximately normal” in terms of level, expressionand/or activity, is meant at least: about 50%, about 60%, about 70%,about 80%, about 90%, and/or about 100%; and/or no more than: about100%, about 120%, about 130%, about 140%, or about 150% of the level,expression or activity of Beta-ENaC in a healthy cell, tissue, or organ.This can be measured using, for example, lung or kidney homogenates, asdescribed in Gambling et al. 2004 Kidney Intl. 65: 1774-1781.Particularly in one embodiment, administration of the appropriate amountof the appropriate Beta-ENaC RNAi agent restores Beta-ENaC level,activity and/or expression and/or Na⁺ reabsorption levels to about 50%to about 150%, more particularly about 60% to about 140%, moreparticularly to about 70% to about 130%, more particularly to about 80%to about 120%, more particularly to about 90% to about 110%, and mostparticularly to about 100% of that of a healthy cell, tissue or organ.The level of Beta-ENaC activity can also be indirectly measured by lungfluid balance. Lung fluid balance can be estimated by calculatingbloodless, wet-to-dry lung weight ratios, which reflect the amount ofextra-vascular lung water. Randrianarison et al. 2007 Am. J. Physiol.Lung Cell. Mol. Physiol. 294: 409-416. The level of Beta-ENaC activitycan also be indirectly measured by histological studies of the lung,particularly the bronchioles, alveolar ducts, alveolar epithelium, andblood vessels. Randrianarison et al. 2007; and Zhou et al. 2008 Am. J.Resp. Crit. Care Med. 178: 1245-1256. Administration of a Beta-ENaC RNAito a patient with a Beta-ENaC-related disease thus particularly restoresthe level, activity, and/or expression of Beta-ENaC and the level of Na⁺reabsorption to an approximately normal level, as determined by directmeasurements of Beta-ENaC mRNA or protein levels, or indirectdeterminations, such as analyses of histological samples or levels oflung fluid.

In addition, in various embodiments, depending on the disease conditionand biological context, it is acceptable to use the RNAi agents of thepresent disclosure to establish a level of Beta-ENaC expression,activity and/or level which is below the normal level, or above thenormal level.

Various factors are known to alter the level of ENaC or, specifically,Beta-ENaC. Hormones that increase the physiological activity of ENaCinclude aldosterone, vasopressin and insulin. Beta-ENaC is specificallyup-regulated by vasopressin and water restriction, as well as duringsodium-bicarbonate loading in rats. These various factors can be used ascontrols in determining the effect of a RNAi agent on Beta-ENaC level.

Types of RNAi Agents and Modification Thereof

The use of RNAi agents or compositions comprising an antisense nucleicacid to down-modulate the expression of a particular protein in a cellis well known in the art. A RNAi agent comprises a sequencecomplementary to, and is capable of hydrogen binding to, the codingstrand of another nucleic acid (e.g., an mRNA). Thus, in variousembodiments, the RNAi agents of the present disclosure encompass anyRNAi agents which target (e.g., are complementary, capable of hydrogenbinding to, etc.) any sequence presented, e.g., in Table 1.

Antisense sequences complementary to an mRNA can be complementary to thecoding region, the 5′ or 3′ untranslated region of the mRNA, and/or aregion bridging the coding and untranslated regions, and/or portionsthereof. Furthermore, a RNAi agent or a portion thereof can becomplementary to a regulatory region of the gene encoding the mRNA, forinstance a transcription or translation initiation sequence orregulatory element. Particularly, a RNAi agent or a portion thereof canbe complementary to a region preceding or spanning the initiation codonon the coding strand or in the 3′ untranslated region of an mRNA.

RNAi agent molecules can be designed according to the rules of Watsonand Crick base pairing. The RNAi agent can be complementary to theentire coding region of Beta-ENaC mRNA, but more particularly is anoligonucleotide which is antisense to only a portion of the coding ornon-coding region of Beta-ENaC mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of Beta-ENaC mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45 or 50 nucleotides in length.

The RNAi agent may have modifications internally, or at one or bothends. The modifications at the ends can help stabilize the RNAi agent,protecting it from degradation by nucleases in the blood. The RNAiagents may optionally be directed to regions of the Beta-ENaC mRNA knownor predicted to be near or at splice sites of the gene; e.g.,exon-intron junctions (as described in, for example, Saxena et al.1998).

The RNAi agents can also optionally be designed to anneal to known orpredicted exposed and/or single-stranded regions of the mRNA (e.g.,loops).

A RNAi agent can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, RNAiagent can be chemically synthesized using naturally-occurringnucleotides or variously modified nucleotides designed to decreaseoff-target effects, and/or increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, the term “ribonucleotide” or “nucleotide” canalso refer to a modified nucleotide or a surrogate replacement moiety.The skilled person is well aware that guanine, cytosine, adenine, anduracil may be replaced by other moieties without substantially alteringthe base pairing properties of an oligonucleotide comprising anucleotide bearing such replacement moiety. For example, withoutlimitation, a nucleotide comprising inosine as its base may base pairwith nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of dsRNA featured in the present disclosure bya nucleotide containing, for example, inosine. In another example,adenine and cytosine anywhere in the oligonucleotide can be replacedwith guanine and uracil, respectively to form G-U Wobble base pairingwith the target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the presentdisclosure.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature (i.e., are naturally occurring), but alsonon-naturally occurring analogs and derivatives of RNA comprising one ormore ribonucleotide/ribonucleoside analogs or derivatives as describedherein or as known in the art. Strictly speaking, a “ribonucleoside”includes a nucleoside base and a ribose sugar, and a “ribonucleotide” isa ribonucleoside with one, two or three phosphate moieties. However, theterms “ribonucleoside” and “ribonucleotide” can be considered to beequivalent as used herein. The RNA can be modified in the nucleobasestructure or in the ribose-phosphate backbone structure, e.g., asdescribed herein below. However, the molecules comprising ribonucleosideanalogs or derivatives must retain the ability to form a duplex. Asnon-limiting examples, an RNA molecule can also include at least onemodified ribonucleoside, including but not limited to a 2′-O-methylmodified nucleotide, a nucleoside comprising a 5′ phosphorothioategroup, a terminal nucleoside linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group, a locked nucleoside, an abasicnucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholinonucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotidemonomer, as described in WO 2008/147824), a phosphoramidate or anon-natural base comprising nucleoside, or any combination thereof.Alternatively, an RNA molecule can comprise at least two modifiedribonucleosides, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20or more, up to the entire length of the dsRNA molecule. Themodifications need not be the same for each of such a plurality ofmodified ribonucleosides in an RNA molecule. In one embodiment, modifiedRNAs contemplated for use in methods and compositions described hereinare peptide nucleic acids (PNAs) that have the ability to form therequired duplex structure and that permit or mediate the specificdegradation of a target RNA via a RISC pathway.

Examples of modified nucleotides which can be used to generate the RNAiagent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

In one embodiment, the present disclosure encompasses modified anymodified variant of any RNAi agent disclosed herein. The modifiedvariant contains the same sequence, but can be modified to containmodifications in the phosphate, sugar, base, nucleotide, etc. Forexample, the modified variant can contain one or more of the modifiednucleotides listed herein, for example a C replaced by a 2′-modified C.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside.In such an instance, a RNAi agent can comprise one or moredeoxynucleosides, including, for example, a deoxynucleoside overhang(s),or one or more deoxynucleosides within the double-stranded portion of adsRNA. However, it is self-evident that under no circumstances is adouble-stranded DNA molecule encompassed by the term “RNAi agent.”

Replacing the 3′-terminal nucleotide overhanging segments of a 21-mersiRNA duplex having two-nucleotide 3′-overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been well tolerated, whereas completesubstitution with deoxyribonucleotides results in no RNAi activity.International PCT Publication No. WO 00/44914, and Beach et al.International PCT Publication No. WO 01/68836 preliminarily suggest thatsiRNA may include modifications to either the phosphate-sugar backboneor the nucleoside to include at least one of a nitrogen or sulfurheteroatom. Kreutzer et al. Canadian Patent Application No. 2,359,180,also describe certain chemical modifications for use in dsRNA constructsin order to counteract activation of double-stranded RNA-dependentprotein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides,and nucleotides containing a 2′-0 or 4′-C methylene bridge. Additional3′-terminal nucleotide overhangs include dT (deoxythimidine),2′-O,4′-C-ethylene thymidine (eT), and 2-hydroxyethyl phosphate (hp).

Parrish et al. 2000 Molecular Cell 6: 1077-1087 tested certain chemicalmodifications targeting the unc-22 gene in C. elegans using long (>25nt) siRNA transcripts. The authors describe the introduction ofthiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

Those skilled in the art will appreciate that it is possible tosynthesize and modify the siRNA as desired, using any conventionalmethod known in the art (see Henschel et al. 2004 DEQOR: a web-basedtool for the design and quality control of siRNAs. Nucleic AcidsResearch 32 (Web Server Issue): W113-W120). Further, it will be apparentto those skilled in the art that there are a variety of regulatorysequences (for example, constitutive or inducible promoters,tissue-specific promoters or functional fragments thereof, etc.) whichare useful for the antisense oligonucleotide, siRNA, or shRNA expressionconstruct/vector.

There are several examples in the art describing sugar, base, phosphateand backbone modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren 1992 TIBS. 17: 34; Usman et al. 1994Nucleic Acids Symp. Ser. 31: 163; Burgin et al. 1996 Biochemistry 35:14090). Sugar modification of nucleic acid molecules are extensivelydescribed in the art.

Additional modifications and conjugations of RNAi agents have beendescribed. Soutschek et al. 2004 Nature 432: 173-178 presentedconjugation of cholesterol to the 3′-end of the sense strand of a siRNAmolecule by means of a pyrrolidine linker, thereby generating a covalentand irreversible conjugate. Chemical modifications (includingconjugation with other molecules) of RNAi agents may also be made toimprove the in vivo pharmacokinetic retention time and efficiency.

In various embodiments, the RNAi agent to Beta-ENaC comprises at leastone 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridineis a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide,wherein the 5′-cytidine is a 2′-modified nucleotide; and/or at least one5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide.

In various embodiments, the RNAi agent comprises a 2′-modificationselected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA).

In another embodiment, the RNAi comprises a gap or missing base. Forexample, the phosphate-sugar backbone may be present, but the basemissing.

In another embodiment, the RNAi agent has a single-stranded nick (e.g.,a break or missing bond in the backbone). In various embodiments, asingle-stranded nick can be in either the sense or anti-sense strand, orboth.

This nick can be, for example, in the sense strand, producing a smallinternally segmented interfering RNA, or sisiRNA, which may have lessoff-target effects than the corresponding RNAi agent without a nick.

The antisense nucleic acid or RNAi agent can also have an alternativebackbone such as locked nucleic acids (LNA), Morpholinos, peptidicnucleic acids (PNA), threose nucleic acid (TNA), or glycol nucleic acid(GNA), and/or it can be labeled (e.g., radiolabeled or otherwisetagged).

One or both strands can comprise an alternative backbone

In yet another embodiment, the RNAi agent employed by the methods of thepresent disclosure can include an α-anomeric nucleic acid molecule. Anα-anomeric nucleic acid molecule forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual β-units, thestrands run parallel to each other. Gaultier et al. 1987 Nucleic Acids.Res. 15: 6625-6641.

The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. 1987 Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987 FEBS Lett.215: 327-330).

In still another embodiment, a RNAi agent is a ribozyme. Ribozymes arecatalytic RNA molecules with ribonuclease activity which are capable ofcleaving a single-stranded nucleic acid, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes [e.g., hammerhead ribozymes(described in Haselhoff et al. 1988, Nature 334: 585-591)] can be usedto catalytically cleave Beta-ENaC mRNA transcripts to thereby inhibittranslation of Beta-ENaC mRNA.

Alternatively, gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of Beta-ENaC (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the Beta-ENaC gene. See generally, Helene 1991Anticancer Drug Des. 6(6): 569-84; Helene et al. 1992 Ann. N.Y. Acad.Sci. 660: 27-36; and Maher 1992, Bioassays 14(12): 807-15.

Production of RNAi Agents

The RNAi agent can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be in anantisense orientation to a target nucleic acid of interest). The RNAiagent can also be produced biologically using an expression vector intowhich a nucleic acid has been subcloned as an shRNA construct (i.e., RNAtranscribed from the inserted nucleic acid will have a first region inan antisense orientation to a target nucleic acid of interest, a secondregion that comprises a loop or hinge, and a third region in a senseorientation to the target nucleic acid of interest, wherein the firstand third regions of the transcript preferably hybridizes with itself,thereby forming a stem-and-loop structure).

Methods of producing RNAi agents are well-known in the art and availableto persons of ordinary skill in the art.

Kits for synthesis of RNAi are commercially available from, e.g., NewEngland Biolabs and Ambion.

Delivery of RNAi Agents

RNAi agents of the present disclosure can be delivered or introduced(e.g., to a cell in vitro, to a test animal, or to a human) by any meansknown in the art.

The RNAi agents of the present disclosure are typically administered toa subject or generated in situ such that they hybridize with cellularmRNA and/or genomic DNA encoding Beta-ENaC, and inhibit expression byinhibiting transcription and/or translation. An example of a route ofadministration of the RNAi agent includes direct injection at a tissuesite. Alternatively, RNAi agents can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing vectors well known in the art and described in, for example,US20070111230, the entire contents of which are incorporated herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

“Introducing into a cell,” when referring to a RNAi agent, meansfacilitating or effecting uptake or absorption into the cell, as isunderstood by those skilled in the art. Absorption or uptake of a RNAiagent can occur through unaided diffusive or active cellular processes,or by auxiliary agents or devices. The meaning of this term is notlimited to cells in vitro; a RNAi agent may also be “introduced into acell,” wherein the cell is part of a living organism. In such aninstance, introduction into the cell will include the delivery to theorganism. For example, for in vivo delivery, a RNAi agent can beinjected into a tissue site or administered systemically. In vivodelivery can also be by a beta-glucan delivery system, such as thosedescribed in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781. In vitro introduction into a cell includesmethods known in the art such as electroporation and lipofection.Further approaches are described herein or known in the art.

Delivery of RNAi agent to tissue is a problem both because the materialmust reach the target organ and must also enter the cytoplasm of targetcells. RNA cannot penetrate cellular membranes, so systemic delivery ofnaked RNAi agent is unlikely to be successful. RNA is quickly degradedby RNAse activity in serum. For these reasons, other mechanisms todeliver RNAi agent to target cells has been devised. Methods known inthe art include but are not limited to: viral delivery (retrovirus,adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine,cationic DOTAP, neutral DOPC) or nanoparticles (cationic polymer, PEI),bacterial delivery (tkRNAi), and also chemical modification (LNA) ofsiRNA to improve stability. Xia et al. 2002 Nat. Biotechnol. 20 andDevroe et al. 2002. BMC Biotechnol. 2 1: 15, disclose incorporation ofsiRNA into a viral vector. Other systems for delivery of RNAi agents arecontemplated and the RNAi agents of the present disclosure can bedelivered by various methods yet to be found and/or approved by the FDAor other regulatory authorities. RNAi agents of the present disclosurecan delivered in a suitable pharmaceutical composition.

Pharmaceutical Compositions of RNAi Agents

As used here, a “pharmaceutical composition” comprises apharmaceutically effective amount of one or more Beta-ENaC RNAi agent, apharmaceutically acceptable carrier, and, optionally, an additionaldisease treatment which works synergistically with the RNAi agent. Asused herein, “pharmacologically effective amount,” “therapeuticallyeffective amount” or simply “effective amount” refers to that amount ofa RNAi agent effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective where there is at least a 10%reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a10% reduction in that parameter. In this embodiment, a therapeuticallyeffective amount of a RNAi agent targeting Beta-ENaC can reduceBeta-ENaC protein levels by at least 10%. In additional embodiments, agiven clinical treatment is considered effective where there is at leasta 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%reduction in a measurable parameter associated with a disease ordisorder, and the therapeutically effective amount of a drug for thetreatment of that disease or disorder is the amount necessary to effectat least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90 or 95% reduction, respectively, in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein.

The pharmaceutical compositions comprising a Beta-ENaC RNAi agent can bein solid form, for example, powders, granules, tablets, pills, gelcaps,gelatin capsules, liposomes, suppositories, chewable forms, or patches.The pharmaceutical compositions comprising a Beta-ENaC RNAi agent canalso be presented in liquid form, for example, solutions, emulsions,suspensions, elixirs, or syrups. Appropriate liquid supports can be, forexample, water, organic solvents such as polyol, such as glycerol orglycols, including propylene glycol and polyethylene glycol, or ethanol,Cremophor EL, or mixtures thereof, in varying proportions, in water. Thecompositions can comprise nano-sized amorphous or crystalline granulescoated with albumin or a surfactant.

Appropriate supports can include, for example, antibacterial andantifungal agents, buffering agents, calcium phosphate, cellulose,methyl cellulose, chlorobutanol, cocoa butter, colorings, dextrin,emulsifiers, enteric coatings, flavorings, gelatin, isotonic agents,lecithin, magnesium stearate, perfuming agents, polyalcohols such asmannitol, injectable organic esters such as ethyl oleate, paraben,phenol sorbic acid, polyethylene glycol, polyvinylpyrrolidine, phosphatebuffered saline (PBS), preserving agents, propylene glycol, sodiumcarboxymethylcellulose, sodium chloride, sorbitol, various sugars(including, but not limited to, sucrose, fructose, galactose, lactoseand trehalose), starch, suppository wax, talc, vegetable oils, such asolive oil and corn oil, vitamins, wax, and/or wetting agents. ForBeta-ENaC RNAi agents, a preferred support comprises dextran and water,e.g. 5% dextrose in water (D5W).

The biologically inert portion of the pharmaceutical composition canoptionally be erodible, allowing timed release of the RNAi agent.

The pharmaceutical composition can comprise additional components whichaid in delivery, stability, efficacy, or reduction of immunogenicity.

Pharmaceutical Composition Comprising a RNAi Agent to Beta-ENaC

Additional components of a pharmaceutical composition comprising a RNAiAgent to Beta-ENaC can be added to aid in delivery, stability, efficacy,or reduction of immunogenicity.

Liposomes have been used previously for drug delivery (e.g., delivery ofa chemotherapeutic). Liposomes (e.g., cationic liposomes) are describedin PCT publications WO02/100435A1, WO03/015757A1, and WO04029213A2; U.S.Pat. Nos. 5,962,016; 5,030,453; and 6,680,068; and U.S. PatentApplication 2004/0208921. A process of making liposomes is alsodescribed in WO04/002453A1. Furthermore, neutral lipids have beenincorporated into cationic liposomes (e.g., Farhood et al. 1995).

Cationic liposomes have been used to deliver RNAi agent to various celltypes (Sioud and Sorensen 2003; U.S. Patent Application 2004/0204377;Duxbury et al., 2004; Donze and Picard, 2002).

Use of neutral liposomes disclosed in Miller et al. 1998, and U.S.Patent Application 2003/0012812.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA or a plasmidfrom which an iRNA is transcribed. SNALPs are described, e.g., in U.S.Patent Application Publication Nos. 20060240093, 20070135372, and inInternational Application No. WO 2009082817.

Chemical transfection using lipid-based, amine-based and polymer-basedtechniques, is disclosed in products from Ambion Inc., Austin, Tex.; andNovagen, EMD Biosciences, Inc, an Affiliate of Merck KGaA, Darmstadt,Germany); Ovcharenko D (2003) “Efficient delivery of siRNAs to humanprimary cells.” Ambion TechNotes 10 (5): 15-16). Additionally, Song etal. (Nat Med. published online (Fete I 0, 2003) doi: 10.1038/nm828) andothers [Caplen et al. 2001 Proc. Natl. Acad. Sci. (USA), 98: 9742-9747;and McCaffrey et al. Nature 414: 34-39] disclose that liver cells can beefficiently transfected by injection of the siRNA into a mammal'scirculatory system.

A variety of molecules have been used for cell-specific RNAi agentdelivery. For example, the nucleic acid-condensing property of protaminehas been combined with specific antibodies to deliver siRNAs. Song etal. 2005 Nat Biotech. 23: 709-717. The self-assembly PEGylatedpolycation polyethylenimine (PEI) has also been used to condense andprotect siRNAs. Schiffelers et al. 2004 Nucl. Acids Res. 32: e149, 141-110.

The siRNA-containing nanoparticles were then successfully delivered tointegrin-overexpressing tumor neovasculature. Hu-Lieskovan et al. 2005Cancer Res. 65: 8984-8992.

The RNAi agents of the present disclosure can be delivered via, forexample, Lipid nanoparticles (LNP); neutral liposomes (NL); polymernanoparticles; double-stranded RNA binding motifs (dsRBMs); or viamodification of the RNAi agent (e.g., covalent attachment to the dsRNA).

Lipid nanoparticles (LNP) are self-assembling cationic lipid basedsystems. These can comprise, for example, a neutral lipid (the liposomebase); a cationic lipid (for siRNA loading); cholesterol (forstabilizing the liposomes); and PEG-lipid (for stabilizing theformulation, charge shielding and extended circulation in thebloodstream).

The cationic lipid can comprise, for example, a headgroup, a linker, atail and a cholesterol tail. The LNP can have, for example, good tumordelivery, extended circulation in the blood, small particles (e.g., lessthan 100 nm), and stability in the tumor microenvironment (which has lowpH and is hypoxic).

Neutral Liposomes (NL) are Non-Cationic Lipid Based Particles.

Polymer nanoparticles are self-assembling polymer-based particles.

Double-stranded RNA binding motifs (dsRBMs) are self-assembling RNAbinding proteins, which will need modifications.

In various embodiments, the RNAi agent to Beta-ENaC is ligated to one ormore diagnostic compound, reporter group, cross-linking agent,nuclease-resistance conferring moiety, natural or unusual nucleobase,lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol,hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

The RNAi agents of the present disclosure can be prepared in apharmaceutical composition comprising various components appropriate forthe particular method of administration of the RNAi agent.

Administration of a RNAi Agent

The pharmaceutical composition comprising a Beta-ENaC can beadministered by buccal, inhalation (including insufflation and deepinhalation), nasal, oral, parenteral, implant, injection or infusion viaepidural, intra-arterial, intra-articular, intra-capsular,intra-cardiac, intra-cerebroventricular, intracranial, intradermal,intramuscular, intra-orbital, intraperitoneal, intra-spinal,intrasternal, intrathecal, intravenous, subarachnoid, sub-capsular,subcutaneous, sub-cuticular, transendothelial, transtracheal,transvascular, rectal, sublingual, topical, and/or vaginal routes. Thismay be by injection, infusion, dermal patch, or any other method knownin the art. The formulation can be powdered, nebulized, aerosolized,granulized or otherwise appropriately prepared for delivery. Theadministration, if liquid, may be slow or via bolus, though, under somecircumstances known in the art, bolus injections may lead to loss ofmaterial through the kidneys.

The pharmaceutical compositions comprising a Beta-ENaC RNAi agent can beadministered with medical devices known in the art. For example, in aparticular embodiment, a RNAi agent can be administered with aneedle-less hypodermic injection device, such as the devices disclosedin U.S. Pat. No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880,4,790,824, or 4,596,556. Examples of well-known implants and modulesuseful in the present disclosure include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medications through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

In certain embodiments, the pharmaceutical compositions comprising aRNAi agent can be formulated to ensure proper distribution in vivo.Administration of a RNAi agent to Beta-ENaC can be systemic (whole-body)or, particularly, targeted to tissues or organs that express (orover-express or demonstrate a hyper-activity of) Beta-ENaC, such aslung, kidney, colon, and glands. Methods for targeting these particulartissues or organs are described herein, and/or are known in the art. Forexample, they can be formulated in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29: 685).

Example targeting moieties include folate or biotin (see, e.g., U.S.Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988)Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman etal. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob.Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe etal. (1995) Am. J. Physiol. 1233: 134), different species of which maycomprise the formulations of the present disclosures, as well ascomponents of the invented molecules; p 120 (Schreier et al. (1994) J.Biol. Chem. 269: 9090); see also K. Keinanen; M. L. Laukkanen (1994)FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

The present disclosure thus encompasses pharmaceutical compositionscomprising one or more RNAi agents to Beta-ENaC, which can optionallycomprise various modifications and/or additional components, for use intreatment of Beta-ENaC-related diseases.

Beta-ENaC-Related Diseases

The present disclosure encompasses RNAi agents to Beta-ENaC andadministration of the RNAi agents to humans and non-human animals totreat various Beta-ENaC-related diseases.

By “Beta-ENaC-related disease” is meant any disease related to adysfunction in the level, expression and/or activity of Beta-ENaC,and/or any disease which can be treated and/or ameliorated by modulatingthe level, expression and/or activity of Beta-ENaC. In particular, itincludes cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1),Liddle's syndrome, hypertension, alkalosis, hypokalemia, andobesity-associated hypertension.

By “cystic fibrosis” or “CF” is meant the common hereditary diseaseassociated with mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene. CFTR encodes a cAMP-dependent Cl—channel and regulates the ENaC. In CF airway epithelia, CFTR-mediatedCl— secretion is defective and ENaC-mediated Na⁺ absorption isincreased. These ion transport defects in CF airways cause airwaysurface liquid (ASL) volume depletion, defective mucus clearance, andmucus adhesion, suggesting that ASL volume depletion is a key mechanismin the pathogenesis of CF lung disease. In experimental mice,airway-specific over-expression of Beta-ENaC demonstrates thataccelerated Na⁺ transport alone is sufficient to produce ASL volumedepletion and CF-like lung disease, including airway mucus obstruction,goblet cell metaplasia, chronic neutrophilic airway inflammation,impaired clearance of bacterial pathogens, and ultimately mortality. SeeZhou et al. 2008, and references cited therein.

By “Liddle's syndrome” is meant an autosomal dominant hereditary form ofhypertension, characterized by an early and severe hypertension, oftenaccompanied by metabolic alkalosis and hypokalemia, all signs that arecharacteristic of an excess of aldosterone (Conn's syndrome).

The plasma levels of aldosterone are low, however. Thus, Liddle'ssyndrome is also called pseudoaldosteronism. This severe form ofhypertension is responsive to treatment with a low-salt diet and Na⁺channel inhibitors (K⁺-sparing diuretics), suggesting a primarydefective regulation of the ENaC. The disease is related to mutations inGamma-ENaC, and also several mutations in Beta-ENaC (P615S, P616L, andY618H in the “PY” motif which has a consensus sequence of PPXY; and alsoR564st, W574st, 579del32, Q589st, T592fr, A593fr, and R595fr, where “fr”is a frameshift, “del” is a deletion, and “st” is a premature stopcodon).

These mutations cause an overexpression of the Na⁺ channels that arehyperactive compared to the wild-type ENaC. The mutations also preventthe downregulation of the channel that normally occurs with a rise inintracellular Na⁺; ENaC channels with the Liddle's mutation remain in ahighly active state despite a high intracellular Na⁺ concentration.Thus, the level and/or activity of a mutated ENaC with Liddle's Syndromecan be modulated by a siRNA to Beta-ENaC, or such a siRNA in combinationwith known treatments for Liddle's syndrome, such as a low-salt diet,and Na⁺ channel inhibitors (K⁺-sparing diuretics).

For additional information on Beta-ENaC-related diseases, see, forexample, Hummler et al. 1999. Am. J. Physiol. Gastrointest. LiverPhysiol. 276: 567-571.

By “obesity-associated hypertension” is meant hypertension related orassociated with obesity, and the like. Obesity is associated withhypertension. Multiple mechanisms have been proposed to explain thiscorrelation, including (in the obese) increased sympathetic activity;increased activity of the renin-angiotensin-aldosterone system;increased cardiac output; and increased mechanical pressure frominterstitial fat around organs, hyperinsulinemia, and/or insulinresistance. Sodium retention by the kidney could result from any ofthese mechanisms. In the connecting tubule and the collecting duct,sodium reabsorption occurs through the ENaC. Levels of Beta-ENaC wereincreased in the kidney in Zucker rats (a model animal for obesity).

Bickel et al. 2001 Am. J. Physiol. Renal Physiol. 281: 639-648. Therelative increases in abundance of this and other sodium transporters,without decreases in the other sodium transporters, likely results inenhanced tubular sodium reabsorption. As a result, these alterations inrenal sodium transporter abundance might play a role in the developmentand/or maintenance of elevated blood pressures in obese mammals,including humans.

By “pseudohypoaldosteronism type 1”, “PHA1”, “PHA-1” and the like ismeant a heterologous clinical syndrome characterized bymineralocorticoid end organ resistance, i.e., urinary loss of Na⁺ andreduced K⁺ excretion despite an elevated level of aldosterone. A severeform of this syndrome is inherited as an autosomal recessive trait,resulting in sometimes lethal episodes of hyponatremia, hypotension, andhyperkalemia, and shows alteration of Na⁺ transport in several organs,kidney, salivary glands, sweat glands, and colon. In several familiesshowing this form of PHA-1, links to mutations in any one of the threeENaC subunits are found (including G37S in Beta-ENaC).

A less severe form of PHA-1 with an autosomal dominant mode ofinheritance is symptomatic mostly during infancy and improves with age.See Hummler et al. 1999. Am. J. Physiol. Gastrointest. Liver Physiol.276: 567-571.

RNAi agents to Beta-ENaC can be used to treat Beta-ENaC-relateddiseases, particularly those diseases associated with alteredexpression, activity and/or levels of Beta-ENaC.

Use of RNAi Agents for Treatment of Beta-ENaC-Related Diseases

The RNAi agents to Beta-ENaC described herein can be formulated intopharmaceutical compositions which can be administered to humans ornon-human animals. These compositions can comprise one or more RNAiagents, and, optionally, additional treatments useful for treatingBeta-ENaC-related diseases. They can be administered as part of anearly/preventative treatment, and can be administered in atherapeutically-effective dosage. The pharmaceutical composition cancomprise a pharmaceutical carrier and can be administered by any methodknown in the art. These various aspects of the present disclosure aredescribed in additional detail below.

RNAi agents to Beta-ENaC can be administered to humans and non-humananimals for treatment of Beta-ENaC-related diseases.

In one embodiment of the present disclosure, the compositions comprisinga Beta-ENaC RNAi agent can be administered to non-human animals. Forexample, the compositions can be given to chickens, turkeys, livestockanimals (such as sheep, pigs, horses, cattle, etc.), companion animals(e.g., cats and dogs) and can have efficacy in treatment of cysticfibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome,hypertension, alkalosis, hypokalemia, and obesity-associatedhypertension and similar diseases. In each case, the RNAi agent toBeta-ENaC would be selected to match the sequence of the Beta-ENaC ofthe genome of the animal, and to, particularly, contain at least 1 ntmismatch from all other genes in that animal's genome. The RNAi agentsof the present disclosure can thus be used in treatment ofBeta-ENaC-related diseases in humans and non-human animals.

As used herein in the context of Beta-ENaC expression, the terms“treat,” “treatment,” and the like, refer to relief from or alleviationof pathological processes mediated by Beta-ENaC expression. In thecontext of the present disclosure insofar as it relates to any of theother conditions recited herein below (other than pathological processesmediated by Beta-ENaC expression), the terms “treat,” “treatment,” andthe like mean to relieve or alleviate at least one symptom associatedwith such condition, or to slow or reverse the progression oranticipated progression of such condition, such as slowing theprogression of a lipid disorder, such as atherosclerosis.

By “treatment” is also meant prophylaxis, therapy, cure, or any otherchange in a patient's condition indicating improvement or absence ofdegradation of physical condition. By “treatment” is meant treatment ofBeta-ENaC-related disease (e.g., cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and obesity-associated hypertension), or anyappropriate treatment of any other ailment the patient has. As usedherein, the terms “treatment” and “treat” refer to both prophylactic andpreventative treatment and curative or disease-modifying treatment,including treatment of patients at risk of contracting a disease orsuspected of having a disease, as well as patients already ill ordiagnosed as suffering from a condition. The terms “treatment” and“treat” also refer to the maintenance and/or promotion of health in anindividual not suffering from a disease but who may be susceptible todeveloping an unhealthy condition, such as nitrogen imbalance or muscleloss. In one embodiment, “treatment” does not encompass prevention of adisease state. Thus, the present disclosure is useful for suppressingexpression of the Beta-ENaC gene and/or treating a Beta-ENaC-relateddisease in an individual afflicted by a Beta-ENaC-related disease, or anindividual susceptible to a Beta-ENaC-related disease. An individual“afflicted” by a Beta-ENaC-related disease has demonstrated detectablesymptoms characteristics of the disease, or had otherwise been shownclinically to have been exposed to or to carry Beta-ENaC-related diseasepathogens or markers. As non-limiting examples, an individual afflictedby a Beta-ENaC-related disease can show outward symptoms; or can show nooutward symptoms but can be shown with a clinical test to carry proteinmarkers associated with a Beta-ENaC-related disease, or proteins orgenetic material associated with a pathogen in the blood.

Early treatment of some Beta-ENaC-related diseases may be moreefficacious if administered early rather than later. Preventative earlyadministration of amiloride (an ENaC inhibitor) was useful in treatingCF model mice, while later administration was not. Similarly, earlyintervention with antimicrobial agents in CF was more effective thantreatment after infection was established. Zhou et al. 2008. Thus, inone particular embodiment, the RNAi agent to Beta-ENaC is administeredearly, prior to disease manifestation, and/or as a preventative agent,rather than administered after disease establishment.

Treatments of Beta-ENaC-related diseases can comprise varioustreatments, comprising a Beta ENaC RNAi agent, and optionally furthercomprising an additional treatment, which can be a method (orprocedure), or an additional composition (e.g., an agent or additionalRNAi agent).

Dosages and Effective Amounts of RNAi Agents

The RNAi agents of the present disclosure are administered in a dosageof a therapeutically effective amount to a patient in need thereof.

An “effective amount” or a “therapeutically effective amount” is anamount that treats a disease or medical condition of an individual, or,more generally, provides a nutritional, physiological or medical benefitto an individual. As used herein, the phrases “therapeutically effectiveamount” and “prophylactically effective amount” refer to an amount thatprovides a therapeutic benefit in the treatment, prevention, ormanagement of pathological processes mediated by Beta-ENaC expression oran overt symptom of pathological processes mediated by Beta-ENaCexpression. The specific amount that is therapeutically effective can bereadily determined by an ordinary medical practitioner, and may varydepending on factors known in the art, such as, for example, the type ofpathological processes mediated by Beta-ENaC expression, the patient'shistory and age, the stage of pathological processes mediated byBeta-ENaC expression, and the administration of other agents thatinhibit pathological processes mediated by Beta-ENaC expression.

In various embodiments of the present disclosure, the patient is atleast about 1, 3, 6, or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55, 60,65, 70, or 75 years of age. In various embodiments, the patient is nomore than about 1, 3, 6, or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55,60, 65, 70, 75, 80, 90, or 100 years of age. In various embodiments thepatient has a body weight of at least about 5, 10, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300,320, 340, 360, 380 or 400 lbs. In various embodiments, the patient has abody weight of no more than about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,360, 380 or 400 lbs.

In various embodiments of the present disclosure, the dosage [measuringonly the active ingredient(s)] can be at least about 1, 5, 10, 25, 50,100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950 or 1000 ng, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000micrograms, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg. In variousembodiments, the dosage can be no more than about 10, 25, 50, 100, 200,250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 mg. In various embodiments, the dosage can be administeredat least more than once a day, daily, more than once a weekly, weekly,bi-weekly, monthly, and/or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12months, or a combination thereof.

In various embodiments, the dosage is correlated to the body weight orbody surface area of the individual. The actual dosage level can bevaried to obtain an amount of active agent which is effective for aparticular patient, composition and mode of administration, withoutbeing toxic to the patient. The selected dose will depend on a varietyof pharmacokinetic factors, including the activity of the particularRNAi agent employed, the route of administration, the rate of excretionof the RNAi agent, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the RNAi agent, the age, sex,weight, condition, general health and prior medical history of thepatient, and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine theeffective amount of the RNAi agent required. A suitable dose will bethat amount which is the lowest dose effective to produce a therapeuticeffect, or a dose low enough to produce a therapeutic effect withoutcausing side effects.

In addition to a therapeutically-effective dosage of one or more RNAiagents to Beta-ENaC, the pharmaceutical compositions of the presentdisclosure can comprise or be used in conjunction with an additionaldisease treatment which works synergistically with the RNAi agent. Forexample, the pharmaceutical composition can comprise an additionalantagonist to ENaC, such as potassium-sparing diuretics, amiloride andtriamterene. Additional treatments can be administered along with thepharmaceutical composition, including, as a non-limiting example,regulation of dietary salt intake. When used to treat cystic fibrosis,the pharmaceutical composition can be used in conjunction with variousmedicaments and therapies known in the art, including, but not limitedto, antibiotics, DNase therapy, albutrol, N-acetylcysteine, breathingtherapy, percussive therapy, aerobic exercise, and various medicamentsand therapies to treat ailments associated with cystic fibrosis (e.g.,diarrhea, osteoporosis, diabetes, bleeding, etc.).

Additional Embodiments of RNAi Agents to Beta-ENaC

In a particular embodiment, the present disclosure encompasses acomposition comprising one or more Beta-ENaC RNAi agents. In oneembodiment, the present disclosure comprises a RNAi agent comprising asense strand and an antisense strand. In one embodiment, the antisensestrand consists of, consists essentially of, or comprises the sequenceof the antisense strand of a RNAi agent listed, e.g., in Table 1. In oneembodiment, the antisense strand consists of, consists essentially of,or comprises a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of any RNAi agent listed, e.g., in Table 1. In oneembodiment, the antisense strand consists of the sequence of theantisense strand of a RNAi agent listed, e.g., in Table 1, and furthercomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In oneembodiment, the antisense strand consists of a sequence with 0, 1, 2, or3 mismatches from that of the antisense strand of a RNAi agent listed,e.g., in Table 1, and further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 nucleotides.

In another embodiment, the composition of the claimed disclosure doesnot comprise any particular individual RNAi agent listed, e.g., inTable 1. In another embodiment of the present disclosure, the RNAi agentto Beta-ENaC does not comprise a sequence of any Beta-ENaC RNAi agentdisclosed in the patent or scientific literature, e.g., U.S. Patent App.No. 60/346,069 (PCT/US02/41850), and Hyde et al. 2009, The 23^(rd) NorthAmerican Cystic Fibrosis Conference, Minneapolis, Oct. 14-17, 2009; orthat available as sc-42418 (and related products) from Santa CruzBiotechnology, Santa Cruz, Calif.

Specific Embodiments of RNAi Agents to Beta-ENaC

Various specific embodiments of a RNAi agent to Beta-ENaC are disclosedherein. Example duplex sequences are provided herein and, e.g., inTable 1. Specific embodiments of the present disclosure include RNAiagents which comprise sequences differing by 0, 1, 2, or 3 nt or by(e.g., with 0, 1, 2 or 3 mismatches) from those of the RNAi agentslisted, e.g., in Table 1.

A mismatch is defined herein as a difference between the base sequenceor length when two sequences are maximally aligned and compared. Amismatch is defined as a position wherein the base of one sequence doesnot match the base of the other sequence. Thus, a mismatch is counted,for example, if a position in one sequence has a particular base (e.g.,A), and the corresponding position on the other sequence has a differentbase (e.g., G).

A mismatch is also counted, e.g., if a position in one sequence has abase (e.g., A), and the corresponding position on the other sequence hasno base (e.g., that position is an abasic nucleotide which comprises aphosphate-sugar backbone but no base). A single-stranded nick in eithersequence (or in the sense or antisense strand) is not counted asmismatch. Thus, as a non-limiting example, no mismatch would be countedif one sequence comprises the sequence AG, but the other sequencecomprises the sequence AG with a single-stranded nick between the A andthe G. A base modification is also not considered a mismatch. Thus, ifone sequence comprises a C, and the other sequence comprises a modifiedC (e.g., 2′-modification) at the same position, no mismatch would becounted.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20807 (SEQ ID NOs: 5 and 6, or SEQ ID NOs:115 and 116).

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20807.

In another particular embodiment, the siRNA comprises AD-20807.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20807.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20826 (SEQ ID NOs: 43 and 44, or SEQ ID NOs:153 and 154).

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20826.

In another particular embodiment, the siRNA comprises AD-20826.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20626.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20832, which comprises SEQ ID NOs: 55 and 56, or SEQ ID NOs:165 and166.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20832.

In another particular embodiment, the siRNA comprises AD-20832.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20832.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20834, which comprises SEQ ID NOs: 59 and 60, or SEQ ID NOs:169 and170.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20834.

In another particular embodiment, the siRNA comprises AD-20834.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20834.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20848, which comprises SEQ ID NOs: 87 and 88, or SEQ ID NOs:197 and198.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20848.

In another particular embodiment, the siRNA comprises AD-20848.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20848.

In one particular embodiment, the present disclosure comprises a RNAiagent comprising a antisense strand comprising at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of:AD-20861, which comprises SEQ ID NOs: 97 and 98, or SEQ ID NOs:207 and208.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20861.

In another particular embodiment, the siRNA comprises AD-20861.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20861.

In one embodiment, the present disclosure comprises a RNAi agentdemonstrating at least about 80% knockdown (no more than about 20%residual gene activity) of the Beta-ENaC gene at an in vitroconcentration of 10 nM in H441 cells.

Thus, in one particular embodiment, the present disclosure comprises aRNAi agent comprising a antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisensestrand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;or AD-20834.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807;AD-20826; AD-20837; AD-20861; or AD-20834.

In another particular embodiment, the siRNA comprises AD-20832;AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; or AD-20834.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; orAD-20834.

In one embodiment, the present disclosure comprises a RNAi agentdemonstrating at least about 70% knockdown (no more than about 30%residual gene activity) of the Beta-ENaC gene at an in vitroconcentration of 10 nM in H441 cells.

Thus, in one particular embodiment, the present disclosure comprises aRNAi agent comprising a antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisensestrand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; or AD-20867.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807;AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865;AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; or AD-20867.

In another particular embodiment, the siRNA comprises AD-20832;AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806;AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; orAD-20867.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; or AD-20867.

In one embodiment, the present disclosure comprises a RNAi agentdemonstrating at least about 60% knockdown (no more than about 40%residual gene activity) of the Beta-ENaC gene at an in vitroconcentration of 10 nM in H441 cells.

Thus, in one particular embodiment, the present disclosure comprises aRNAi agent comprising a antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisensestrand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831;AD-20862; AD-20808; or AD-20827.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807;AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865;AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813;AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; or AD-20827.

In another particular embodiment, the siRNA comprises: AD-20832;AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806;AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825;AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; orAD-20827.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831;AD-20862; AD-20808; or AD-20827.

In one embodiment, the present disclosure comprises a RNAi agentdemonstrating at least about 50% knockdown (no more than about 50%residual gene activity) of the Beta-ENaC gene at an in vitroconcentration of 10 nM in H441 cells.

Thus, in one particular embodiment, the present disclosure comprises aRNAi agent comprising a antisense strand comprising at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisensestrand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831;AD-20862; AD-20808; AD-20827; AD-20828; AD-20812; AD-20836; or AD-20822.

In another particular embodiment, the siRNA also further comprises asense strand comprising at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807;AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865;AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813;AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; AD-20827; AD-20828;AD-20812; AD-20836; or AD-20822.

In another particular embodiment, the siRNA comprises AD-20832;AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806;AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825;AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808;AD-20827; AD-20828; AD-20812; AD-20836; or AD-20822.

In another particular embodiment, the siRNA has a sequence consisting ofthat of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861;AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839;AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831;AD-20862; AD-20808; AD-20827; AD-20828; AD-20812; AD-20836; or AD-20822.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20805.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20806.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20807.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20808.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20809.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20810.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20811.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20812.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20813.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20814.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20815.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20816.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20817.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20818.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20819.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20820.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20821.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20822.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20823.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20824.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20825.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20826.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20827.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20828.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20829.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20830.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20831.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20832.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20833.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20834.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20835.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20836.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20837.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20838.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20839.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20840.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20841.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20842.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20843.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20844.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20845.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20846.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20847.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20848.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20849.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20850.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20851.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20852.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20861.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20862.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20863.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20864.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20865.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20866.

In one embodiment, the present disclosure comprises a RNAi agentcomprising a sense strand comprising at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the sense strand and/or an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand of AD-20867.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20805, which comprises SEQ ID NOs. 111-112, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20806, which comprises SEQ ID NOs. 113-114, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20807, which comprises SEQ ID NOs. 115-116, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20808, which comprises SEQ ID NOs. 117-118, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20809, which comprises SEQ ID NOs. 119-120, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20810, which comprises SEQ ID NOs. 121-122, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20811, which comprises SEQ ID NOs. 123-124, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20812, which comprises SEQ ID NOs. 125-126, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20813, which comprises SEQ ID NOs. 127-128, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20814, which comprises SEQ ID NOs. 129-130, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20815, which comprises SEQ ID NOs. 131-132, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20816, which comprises SEQ ID NOs. 133-134, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20817, which comprises SEQ ID NOs. 135-136, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20818, which comprises SEQ ID NOs. 137-138, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20819, which comprises SEQ ID NOs. 139-140, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20820, which comprises SEQ ID NOs. 141-142, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20821, which comprises SEQ ID NOs. 143-144, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20822, which comprises SEQ ID NOs. 145-146, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20823, which comprises SEQ ID NOs. 147-148, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20824, which comprises SEQ ID NOs. 149-150, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20825, which comprises SEQ ID NOs. 151-152, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20826, which comprises SEQ ID NOs. 153-154, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20827, which comprises SEQ ID NOs. 155-156, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20828, which comprises SEQ ID NOs. 157-158, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20829, which comprises SEQ ID NOs. 159-160, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20830, which comprises SEQ ID NOs. 161-162, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20831, which comprises SEQ ID NOs. 163-164, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20832, which comprises SEQ ID NOs. 165-166, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20833, which comprises SEQ ID NOs. 167-168, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20834, which comprises SEQ ID NOs. 169-170, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20835, which comprises SEQ ID NOs. 171-172, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20836, which comprises SEQ ID NOs. 173-174, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20837, which comprises SEQ ID NOs. 175-176, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20838, which comprises SEQ ID NOs. 177-178, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20839, which comprises SEQ ID NOs. 179-180, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20840, which comprises SEQ ID NOs. 181-182, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20841, which comprises SEQ ID NOs. 183-184, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20842, which comprises SEQ ID NOs. 185-186, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20843, which comprises SEQ ID NOs. 187-188, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20844, which comprises SEQ ID NOs. 189-190, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20845, which comprises SEQ ID NOs. 191-192, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20846, which comprises SEQ ID NOs. 193-194, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20847, which comprises SEQ ID NOs. 195-196, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20848, which comprises SEQ ID NOs. 197-198, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20849, which comprises SEQ ID NOs. 199-200, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20850, which comprises SEQ ID NOs. 201-202, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20851, which comprises SEQ ID NOs. 203-204, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20852, which comprises SEQ ID NOs. 205-206, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20861, which comprises SEQ ID NOs. 207-208, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20862, which comprises SEQ ID NOs. 209-210, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20863, which comprises SEQ ID NOs. 211-212, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20864, which comprises SEQ ID NOs. 213-214, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20865, which comprises SEQ ID NOs. 215-216, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20866, which comprises SEQ ID NOs. 217-218, and modifiedvariants thereof.

In one embodiment, the present disclosure relates to a compositioncomprising a RNAi agent comprising a sense strand and an antisensestrand, wherein the antisense strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nucleotides from the antisensestrand of: AD-20867, which comprises SEQ ID NOs. 219-220, and modifiedvariants thereof.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20805, which comprises SEQ ID NOs. 111and 112.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20806, which comprises SEQ ID NOs. 113and 114

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20807, which comprises SEQ ID NOs. 115and 116.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20808, which comprises SEQ ID NOs. 117and 118.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20809, which comprises SEQ ID NOs. 119and 120.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20810, which comprises SEQ ID NOs. 121and 122.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20811, which comprises SEQ ID NOs. 123and 124.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20812, which comprises SEQ ID NOs. 125and 126.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20813, which comprises SEQ ID NOs. 127and 128.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20814, which comprises SEQ ID NOs. 129and 130.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20815, which comprises SEQ ID NOs. 131and 132.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20816, which comprises SEQ ID NOs. 133and 134.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20817, which comprises SEQ ID NOs. 135and 136.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20818, which comprises SEQ ID NOs. 137and 138.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20819, which comprises SEQ ID NOs. 139and 140.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20820, which comprises SEQ ID NOs. 141and 142.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20821, which comprises SEQ ID NOs. 143and 144.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20822, which comprises SEQ ID NOs. 145and 146.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20823, which comprises SEQ ID NOs. 147and 148.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20824, which comprises SEQ ID NOs. 149and 150.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20825, which comprises SEQ ID NOs. 151and 152.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20826, which comprises SEQ ID NOs. 153and 154.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20827, which comprises SEQ ID NOs. 155and 156.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20828, which comprises SEQ ID NOs. 157and 158.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20829, which comprises SEQ ID NOs. 159and 160.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20830, which comprises SEQ ID NOs. 161and 162.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20831, which comprises SEQ ID NOs. 163and 164.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20832, which comprises SEQ ID NOs. 165and 166.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20833, which comprises SEQ ID NOs. 167and 168.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20834, which comprises SEQ ID NOs. 169and 170.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20835, which comprises SEQ ID NOs. 171and 172.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20836, which comprises SEQ ID NOs. 173and 174.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20837, which comprises SEQ ID NOs. 175and 176.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20838, which comprises SEQ ID NOs. 177and 178.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20839, which comprises SEQ ID NOs. 179and 180.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20840, which comprises SEQ ID NOs. 181and 182.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20841, which comprises SEQ ID NOs. 183and 184.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20842, which comprises SEQ ID NOs. 185and 186.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20843, which comprises SEQ ID NOs. 187and 188.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20844, which comprises SEQ ID NOs. 189and 190.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20845, which comprises SEQ ID NOs. 191and 192.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20846, which comprises SEQ ID NOs. 193and 194.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20847, which comprises SEQ ID NOs. 195and 196.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20848, which comprises SEQ ID NOs. 197and 198.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20849, which comprises SEQ ID NOs. 199and 200.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20850, which comprises SEQ ID NOs. 201and 202.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20851, which comprises SEQ ID NOs. 203and 204.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20852, which comprises SEQ ID NOs. 205and 206.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20861, which comprises SEQ ID NOs. 207and 208.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20862, which comprises SEQ ID NOs. 209and 210.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20863, which comprises SEQ ID NOs. 211and 212.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20864, which comprises SEQ ID NOs. 213and 214.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20865, which comprises SEQ ID NOs. 215and 216.

In one embodiment, the composition comprises a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20866, which comprises SEQ ID NOs. 217and 218.

and In one embodiment, the composition comprises a RNAi agent comprisinga sense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0 nucleotidesfrom the antisense strand of: AD-20867, which comprises SEQ ID NOs. 219and 220.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20805, which comprises SEQ ID NOs. 1 and 2.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20806, which comprises SEQ ID NOs. 3 and 4.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20807, which comprises SEQ ID NOs. 5 and 6.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20808, which comprises SEQ ID NOs. 7 and 8.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20809, which comprises SEQ ID NOs. 9 and 10.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20810, which comprises SEQ ID NOs. 11 and 12.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20811, which comprises SEQ ID NOs. 13 and 14.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20812, which comprises SEQ ID NOs. 15 and 16.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20813, which comprises SEQ ID NOs. 17 and 18.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20814, which comprises SEQ ID NOs. 19 and 20.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20815, which comprises SEQ ID NOs. 21 and 22.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20816, which comprises SEQ ID NOs. 23 and 24.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20817, which comprises SEQ ID NOs. 25 and 26.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20818, which comprises SEQ ID NOs. 27 and 28.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20819, which comprises SEQ ID NOs. 29 and 30.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20820, which comprises SEQ ID NOs. 31 and 32.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20821, which comprises SEQ ID NOs. 33 and 34.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20822, which comprises SEQ ID NOs. 35 and 36.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20823, which comprises SEQ ID NOs. 37 and 38.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20824, which comprises SEQ ID NOs. 39 and 40.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20825, which comprises SEQ ID NOs. 41 and 42.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20826, which comprises SEQ ID NOs. 43 and 44.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20827, which comprises SEQ ID NOs. 45 and 46.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20828, which comprises SEQ ID NOs. 47 and 48.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20829, which comprises SEQ ID NOs. 49 and 50.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20830, which comprises SEQ ID NOs. 51 and 52.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20831, which comprises SEQ ID NOs. 53 and 54.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20832, which comprises SEQ ID NOs. 55 and 56.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20833, which comprises SEQ ID NOs. 57 and 58.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20834, which comprises SEQ ID NOs. 59 and 60.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20835, which comprises SEQ ID NOs. 61 and 62.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20836, which comprises SEQ ID NOs. 63 and 64.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20837, which comprises SEQ ID NOs. 65 and 66.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20838, which comprises SEQ ID NOs. 67 and 68.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20839, which comprises SEQ ID NOs. 69 and 70.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20840, which comprises SEQ ID NOs. 71 and 72.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20841, which comprises SEQ ID NOs. 73 and 74.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20842, which comprises SEQ ID NOs. 75 and 76.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20843, which comprises SEQ ID NOs. 77 and 78.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20844, which comprises SEQ ID NOs. 79 and 80.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20845, which comprises SEQ ID NOs. 81 and 82.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20846, which comprises SEQ ID NOs. 83 and 84.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20847, which comprises SEQ ID NOs. 85 and 86.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20848, which comprises SEQ ID NOs. 87 and 88.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20849, which comprises SEQ ID NOs. 89 and 90.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20850, which comprises SEQ ID NOs. 91 and 92.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20851, which comprises SEQ ID NOs. 93 and 94.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20852, which comprises SEQ ID NOs. 95 and 96.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20861, which comprises SEQ ID NOs. 97 and 98.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20862, which comprises SEQ ID NOs. 99 and 100.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20863, which comprises SEQ ID NOs. 101 and102.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20864, which comprises SEQ ID NOs. 103 and104.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20865, which comprises SEQ ID NOs. 105 and106.

In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20866, which comprises SEQ ID NOs. 107 and108.

and In one embodiment, the composition comprises a modified variant of aRNAi agent, wherein the variant comprises a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense sequence of: AD-20867, which comprises SEQ ID NOs. 109 and110.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the present disclosure comprises AD-20805 (SEQ IDNOs: 1 and 2, or SEQ ID NOs: 111 and 112).

In one embodiment, the present disclosure comprises AD-20806 (SEQ IDNOs: 3 and 4, or SEQ ID NOs: 113 and 114).

In one embodiment, the present disclosure comprises AD-20807 (SEQ IDNOs: 5 and 6, or SEQ ID NOs: 115 and 116).

In one embodiment, the present disclosure comprises AD-20808 (SEQ IDNOs: 7 and 8, or SEQ ID NOs: 117 and 118).

In one embodiment, the present disclosure comprises AD-20809 (SEQ IDNOs: 9 and 10, or SEQ ID NOs: 119 and 120).

In one embodiment, the present disclosure comprises AD-20810 (SEQ IDNOs: 11 and 12, or SEQ ID NOs: 121 and 122).

In one embodiment, the present disclosure comprises AD-20811 (SEQ IDNOs: 13 and 14, or SEQ ID NOs: 123 and 124).

In one embodiment, the present disclosure comprises AD-20812 (SEQ IDNOs: 15 and 16, or SEQ ID NOs: 125 and 126).

In one embodiment, the present disclosure comprises AD-20813 (SEQ IDNOs: 17 and 18, or SEQ ID NOs: 127 and 128).

In one embodiment, the present disclosure comprises AD-20814 (SEQ IDNOs: 19 and 20, or SEQ ID NOs: 129 and 130).

In one embodiment, the present disclosure comprises AD-20815 (SEQ IDNOs: 21 and 22, or SEQ ID NOs: 131 and 132).

In one embodiment, the present disclosure comprises AD-20816 (SEQ IDNOs: 23 and 24, or SEQ ID NOs: 133 and 134).

In one embodiment, the present disclosure comprises AD-20817 (SEQ IDNOs: 25 and 26, or SEQ ID NOs: 135 and 136).

In one embodiment, the present disclosure comprises AD-20818 (SEQ IDNOs: 27 and 28, or SEQ ID NOs: 137 and 138).

In one embodiment, the present disclosure comprises AD-20819 (SEQ IDNOs: 29 and 30, or SEQ ID NOs: 139 and 140).

In one embodiment, the present disclosure comprises AD-20820 (SEQ IDNOs: 31 and 32, or SEQ ID NOs: 141 and 142).

In one embodiment, the present disclosure comprises AD-20821 (SEQ IDNOs: 33 and 34, or SEQ ID NOs: 143 and 144).

In one embodiment, the present disclosure comprises AD-20822 (SEQ IDNOs: 35 and 36, or SEQ ID NOs: 145 and 146).

In one embodiment, the present disclosure comprises AD-20823 (SEQ IDNOs: 37 and 38, or SEQ ID NOs: 147 and 148).

In one embodiment, the present disclosure comprises AD-20824 (SEQ IDNOs: 39 and 40, or SEQ ID NOs: 149 and 150).

In one embodiment, the present disclosure comprises AD-20825 (SEQ IDNOs: 41 and 42, or SEQ ID NOs: 151 and 152).

In one embodiment, the present disclosure comprises AD-20826 (SEQ IDNOs: 43 and 44, or SEQ ID NOs: 153 and 154).

In one embodiment, the present disclosure comprises AD-20827 (SEQ IDNOs: 45 and 46, or SEQ ID NOs: 155 and 156).

In one embodiment, the present disclosure comprises AD-20828 (SEQ IDNOs: 47 and 48, or SEQ ID NOs: 157 and 158).

In one embodiment, the present disclosure comprises AD-20829 (SEQ IDNOs: 49 and 50, or SEQ ID NOs: 159 and 160).

In one embodiment, the present disclosure comprises AD-20830 (SEQ IDNOs: 51 and 52, or SEQ ID NOs: 161 and 162).

In one embodiment, the present disclosure comprises AD-20831 (SEQ IDNOs: 53 and 54, or SEQ ID NOs: 163 and 164).

In one embodiment, the present disclosure comprises AD-20832 (SEQ IDNOs: 55 and 56, or SEQ ID NOs: 165 and 166).

In one embodiment, the present disclosure comprises AD-20833 (SEQ IDNOs: 57 and 58, or SEQ ID NOs: 167 and 168).

In one embodiment, the present disclosure comprises AD-20834 (SEQ IDNOs: 59 and 60, or SEQ ID NOs: 169 and 170).

In one embodiment, the present disclosure comprises AD-20835 (SEQ IDNOs: 61 and 62, or SEQ ID NOs: 171 and 172).

In one embodiment, the present disclosure comprises AD-20836 (SEQ IDNOs: 63 and 64, or SEQ ID NOs: 173 and 174).

In one embodiment, the present disclosure comprises AD-20837 (SEQ IDNOs: 65 and 66, or SEQ ID NOs: 175 and 176).

In one embodiment, the present disclosure comprises AD-20838 (SEQ IDNOs: 67 and 68, or SEQ ID NOs: 177 and 178).

In one embodiment, the present disclosure comprises AD-20839 (SEQ IDNOs: 69 and 70, or SEQ ID NOs: 179 and 180).

In one embodiment, the present disclosure comprises AD-20840 (SEQ IDNOs: 71 and 72, or SEQ ID NOs: 181 and 182).

In one embodiment, the present disclosure comprises AD-20841 (SEQ IDNOs: 73 and 74, or SEQ ID NOs: 183 and 184).

In one embodiment, the present disclosure comprises AD-20842 (SEQ IDNOs: 75 and 76, or SEQ ID NOs: 185 and 186).

In one embodiment, the present disclosure comprises AD-20843 (SEQ IDNOs: 77 and 78, or SEQ ID NOs: 187 and 188).

In one embodiment, the present disclosure comprises AD-20844 (SEQ IDNOs: 79 and 80, or SEQ ID NOs: 189 and 190).

In one embodiment, the present disclosure comprises AD-20845 (SEQ IDNOs: 81 and 82, or SEQ ID NOs: 191 and 192).

In one embodiment, the present disclosure comprises AD-20846 (SEQ IDNOs: 83 and 84, or SEQ ID NOs: 193 and 194).

In one embodiment, the present disclosure comprises AD-20847 (SEQ IDNOs: 85 and 86, or SEQ ID NOs: 195 and 196).

In one embodiment, the present disclosure comprises AD-20848 (SEQ IDNOs: 87 and 88, or SEQ ID NOs: 197 and 198).

In one embodiment, the present disclosure comprises AD-20849 (SEQ IDNOs: 89 and 90, or SEQ ID NOs: 199 and 200).

In one embodiment, the present disclosure comprises AD-20850 (SEQ IDNOs: 91 and 92, or SEQ ID NOs: 201 and 202).

In one embodiment, the present disclosure comprises AD-20851 (SEQ IDNOs: 93 and 94, or SEQ ID NOs: 203 and 204).

In one embodiment, the present disclosure comprises AD-20852 (SEQ IDNOs: 95 and 96, or SEQ ID NOs: 205 and 206).

In one embodiment, the present disclosure comprises AD-20861 (SEQ IDNOs: 97 and 98, or SEQ ID NOs: 207 and 208).

In one embodiment, the present disclosure comprises AD-20862 (SEQ IDNOs: 99 and 100, or SEQ ID NOs: 209 and 210).

In one embodiment, the present disclosure comprises AD-20863 (SEQ IDNOs: 101 and 102, or SEQ ID NOs: 211 and 212).

In one embodiment, the present disclosure comprises AD-20864 (SEQ IDNOs: 103 and 104, or SEQ ID NOs: 213 and 214).

In one embodiment, the present disclosure comprises AD-20865 (SEQ IDNOs: 105 and 106, or SEQ ID NOs: 215 and 216).

In one embodiment, the present disclosure comprises AD-20866 (SEQ IDNOs: 107 and 108, or SEQ ID NOs: 217 and 218).

In one embodiment, the present disclosure comprises AD-20867 (SEQ IDNOs: 109 and 110, or SEQ ID NOs: 219 and 220).

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20805.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20806.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20807.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20808.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20809.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20810.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20811.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20812.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20813.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20814.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20815.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20816.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20817.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20818.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20819.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20820.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20821.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20822.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20823.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20824.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20825.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20826.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20827.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20828.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20829.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20830.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20831.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20832.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20833.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20834.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20835.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20836.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20837.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20838.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20839.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20840.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20841.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20842.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20843.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20844.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20845.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20846.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20847.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20848.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20849.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20850.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20851.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20852.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20861.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20862.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20863.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20864.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20865.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20866.

In one embodiment, the RNAi agent comprises an antisense strand that isthe exact sequence and length of the antisense strand of AD-20867. Inthese various embodiments, a RNAi agent comprising an antisense strandthat is the exact sequence and length of a recited antisense strand of arecited RNAi agent can comprise modified nucleotides, 3′-end caps,and/or other modifications which do not alter the sequence or length ofthe RNAi agent.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20805.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20806.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20807.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20808.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20809.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20810.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20811.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20812.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20813.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20814.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20815.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20816.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20817.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20818.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20819.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20820.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20821.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20822.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20823.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20824.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20825.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20826.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20827.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20828.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20829.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20830.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20831.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20832.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20833.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20834.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20835.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20836.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20837.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20838.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20839.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20840.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20841.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20842.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20843.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20844.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20845.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20846.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20847.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20848.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20849.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20850.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20851.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20852.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20861.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20862.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20863.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20864.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20865.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20866.

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20867.

Various Embodiments of a RNAi Agent to Beta-ENaC

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20805, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof, e.g., 0-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3,2-4, or 2-5 nt, etc.).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20806, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20807, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20808, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20809, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20810, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20811, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20812, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20813, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20814, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20815, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20816, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20817, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20818, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20819, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20820, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20821, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20822, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20823, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20824, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20825, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20826, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20827, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20828, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20829, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20830, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20831, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20832, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20833, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20834, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20835, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20836, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20837, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20838, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20839, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20840, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20841, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20842, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20843, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20844, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20845, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20846, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20847, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20848, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20849, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20850, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20851, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20852, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20861, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20862, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20863, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20864, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20865, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20866, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

In one embodiment, the RNAi agent comprises an antisense strandconsisting of a sequence with 0, 1, 2, or 3 mismatches from that of theantisense strand of AD-20867, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any rangethereof).

Additional Particular Specific Embodiments

In various embodiments, the disclosure comprises a RNAi agent comprisinga sense and an antisense strand, wherein the antisense strand comprisesat least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt fromthe antisense strand of any RNAi agent disclosed herein.

Thus, in various embodiments:

The disclosure comprises a RNAi agent comprising a sense and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisensestrand of any one or more of the following duplexes, or modified orunmodified variants thereof: AD-20832; AD-20848; AD-20807; AD-20826;AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809,AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816,AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823,AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831,AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841,AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849,AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865,AD-20866, AD-20867, or modified or unmodified variants thereof.

Additional Particular Specific Embodiments

In various embodiments, the disclosure comprises a RNAi agent comprisinga first and a second strand, wherein the sequence of the first strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from the sequence of the first strand of, and the sequence of thesecond strand comprises at least 15 contiguous nucleotides differing by0, 1, 2, or 3 nucleotides from the sequence of the second strand, of anyRNAi agent, disclosed herein.

Thus, in various embodiments:

The disclosure comprises a RNAi agent comprising a sense and anantisense strand, wherein the sequence of the first strand comprises atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from thesequence of the first strand of, and the sequence of the second strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the sequence of the second strand, of any one or moreof the following duplexes, or modified or unmodified variants thereof:AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834,AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD-20811, AD-20812,AD-20813, AD-20814, AD-20815, AD-20816, AD-20817, AD-20818, AD-20819,AD-20820, AD-20821, AD-20822, AD-20823, AD-20824, AD-20825, AD-20827,AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836,AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844,AD-20845, AD-20846, AD-20847, AD-20849, AD-20850, AD-20851, AD-20852,AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867, or modifiedor unmodified variants thereof.

Additional Particular Embodiments

In various embodiments, the disclosure comprises a RNAi agent comprisinga sense and an antisense strand, wherein the antisense strand comprisesor consists of the antisense strand of any RNAi agent disclosed herein.

Thus, the following are provided as examples of the various embodiments.

The disclosure comprises a RNAi agent comprising a sense and anantisense strand, wherein the antisense strand comprises or consists ofthe antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826;AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809,AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816,AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823,AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831,AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841,AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849,AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865,AD-20866, AD-20867, or modified or unmodified variants thereof.

In various embodiments, the disclosure comprises a RNAi agent comprisinga sense and an antisense strand, wherein the antisense strand comprisesat least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt fromthe antisense strand of any RNAi agent disclosed herein, or modified orunmodified variants thereof, wherein the antisense strand optionallyfurther comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nt (or anyrange thereof, e.g., 0-1, 1-2, 1-3, 1-4 nt, etc.).

Thus, in various embodiments, the disclosure comprises a RNAi agentcomprising a sense and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nt from the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826;AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809,AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816,AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823,AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831,AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841,AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849,AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865,AD-20866, AD-20867, or modified or unmodified variants thereof, whereinthe antisense strand optionally further comprises 0, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 or more nt (or any range thereof, e.g., 0-1, 1-2, 1-3,1-4 nt, etc.).

In various embodiments, the disclosure comprises a RNAi agent comprisinga first and a second strand, wherein the sequence of the first strandcomprises or consists of the sequence of the first strand of, and thesequence of the second strand comprises or consists of the sequence ofthe second strand of any RNAi agent disclosed herein, or modified orunmodified variants thereof.

Thus, in various embodiments, the disclosure comprises a RNAi agentcomprising a first and a second strand, wherein the sequence of thefirst strand comprises or consists of the sequence of the first strandof, and the sequence of the second strand comprises or consists of thesequence of the second strand of: AD-20832; AD-20848; AD-20807;AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808,AD-20809, AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815,AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822,AD-20823, AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830,AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840,AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847,AD-20849, AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864,AD-20865, AD-20866, AD-20867.

In one embodiment, the disclosure comprises any one or more RNAi agentlisted herein.

Overlapping Sets of RNAi Agents to Beta-ENaC

In various embodiments, the present disclosure relates to groups of RNAiagents to Beta-ENaC with overlapping sequences. Thus, the presentdisclosure encompasses groups of RNAi agents wherein each RNAi agent inthe group overlaps with each other RNAi agent in the same group by atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or morenucleotides. Particularly, in one embodiment, the overlap is at least 12nt.

Some of the RNAi agents listed herein overlap each other in sequence.Table 2 presents a compilation of some of these groups of overlappingRNAi agents, wherein each member of a group overlaps with each othermember of the same group by at least 12 nt. A 12-nt portion of theoverlap of the sense and anti-sense strand are presented.

Thus, for example, as shown in Table 2, the sequences of RNAi agentsAD-20807 and AD-20832 overlap, wherein the overlap in the sense strandcomprises the sequence UGAAGAAGUACC (SEQ ID NO: 223); these RNAi agentsalso overlap in the anti-sense strand sequence, wherein the overlapcomprises the sequence GGUACUUCUUCA (SEQ ID NO: 224). The RNAi agentsAD-20807, AD-20862 and AD-20832 all overlap in the sense strand, whereinthe overlap comprises the sequence GAAGAAGUACCU (SEQ ID NO: 225); theseRNAi agents also overlap in the anti-sense strand, wherein the overlapcomprises the sequence AGGUACUUCUUC (SEQ ID NO: 226). Thus, these andother various sets of overlapping RNAi agents presented in Table 2 sharecommon technical features, for example, the overlap in the sense andanti-sense strand.

Particular sets of overlapping RNAi agents to Beta-ENaC are providedbelow in Table 2.

The present disclosure thus encompasses any group or subgroup of RNAiagents comprising a common technical feature, wherein the commontechnical feature is an overlap (e.g., of at least 12 nt) of a sequencein the sense or anti-sense strand.

Thus:

The present disclosure encompasses a RNAi agent comprising: an antisensestrand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand, and/or a sense strand comprisingat least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt fromthe sense strand, of any of the group of: AD-20807 and AD-20832 (or anyother group presented in Table 2).

The present disclosure encompasses a RNAi agent comprising a first and asecond strand, wherein the first strand comprises at least 15 contiguousnucleotides differing by 0, 1, 2, or 3 nt from the first strand of,and/or the second strand comprises at least 15 contiguous nucleotidesdiffering by 0, 1, 2, or 3 nt from the second strand, of any of thegroup of: AD-20807 and AD-20832 (or any other group presented in Table2).

The present disclosure encompasses a RNAi agent comprising a first and asecond strand, wherein the first strand comprises or consists of thesequence of a first strand of, and/or the second strand comprises orconsists of the sequence of, any of the group of: AD-20807 and AD-20832(or any other group presented in Table 2).

The disclose encompasses a RNAi agent comprising a first and a secondstrand (wherein the first and second strand may optionally be covalentlylinked, linked via a loop or linker, or contiguous), and wherein thefirst and/or second strand comprise, consist essentially of, or consistof sequences with 0, 1, 2, or 3 nt or by mismatches of any of the groupof: AD-20807 and AD-20832 (or any other group presented in Table 2),optionally further comprising 0-10 nt or bp.

The present disclosure similarly encompasses various embodimentsencompassing groups of overlapping RNAi agents presented in Table 2.

Additional Definitions

The articles “a” and “an” as used herein and in the claims refer to oneor more than one (at least one) of the grammatical object of thearticle.

The terms “RNAi agent,” “RNAi agents”, “RNAi agent(s)” and the like allrefer without limitation to one or more RNAi agents of the presentdisclosure.

The designations of particular example duplexes of RNAi agents toBeta-ENaC disclosed herein on occasion have the suffix “b” followed by anumber. This indicates a batch number. Thus, the suffix “b1” indicates“batch 1.” Thus, a RNAi duplex designated, for example, “AD-20807-b1” isspecifically from batch 1 and has the same sequence as any RNAi agentdesignated “AD-20807”.

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the present disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein.

Claims to the present disclosure are non-limiting and are providedbelow.

Although particular embodiments and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims, or the scope of subject matter ofclaims of any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the present disclosure withoutdeparting from the spirit and scope of the present disclosure as definedby the claims. The choice of nucleic acid starting material, clone ofinterest, or library type is believed to be a matter of routine for aperson of ordinary skill in the art with knowledge of the embodimentsdescribed herein. Other aspects, advantages, and modificationsconsidered to be within the scope of the following claims. Redrafting ofclaim scope in later-filed corresponding applications may be due tolimitations by the patent laws of various countries and should not beinterpreted as giving up subject matter of the claims.

Various additional formulations and obvious variants of the describedRNAi agents to Beta-ENaC can be devised by those of ordinary skill inthe art. Non-limiting example RNAi agents to Beta-ENaC are described inthe Examples below, which do not limit the scope of the presentdisclosure as described in the claims.

EXAMPLES Example 1 Bioinformatics and Beta-ENaC RNAi Agent (siRNA)Sequences

Beta-ENaC oligonucleotide design is carried out to identify siRNAstargeting mRNAs encoding the Beta-ENaC gene [“sodium channel,nonvoltage-gated 1 beta” from human (NCBI human symbol SCNN1B) and theorthologous sequences from cynomolgus monkey (Macaca fascicularis) andrat (Rattus norvegicus)]. The design process uses the SCNNB1 transcriptsNM_(—)000336.2 from human (NCBI GeneId 6338), NM_(—)012648.1 from rat(NCBI GeneId 24767), and a full length cynomolgus monkey sequence(described herein).

All siRNA duplexes are designed to have 100% identity to all threeSCNNB1 transcripts. All sequences are from Transcript NM_(—)000336.

Unmodified and modified sequences are listed in Table 1. Unmodifiedsequences include both the sense and antisense sequences which arelisted as SEQ ID NO: 111 to 220. The relative positions of the firstresidue as compared to the human Beta-ENaC transcript in SEQ ID NO: 222are also provided.

As described below, Table 1 also provides example modified variants ofthese sequences (SEQ ID NO: 1 to 110). For the Table 1 columns, “S”represents the sense strand, “AS” represents the antisense strand, and“Pos'n” represents the position of the first nucleotide. Modifiednucleotides, as indicated by lower case letters (e.g., “c” and “u”) areas described in Table 1A, below.

In the sequences in Table 1, the modified and unmodified sequences canoptionally comprise the sequence “dTsdT” at the 3′ end. Thus, forexample, AD-20805 can optionally have the modified sequencecAGuGAcuAcAAcAcGAccdTsdT (SEQ ID NO: 429) in the sense strand andGGUCGUGUUGuAGUcACUGdTsdT (SEQ ID NO: 430) in the anti-sense strand. Asnoted in Table 1A, below, dT is 2′-deoxy-thymidine-5′-phosphate and sdTis 2′-deoxy Thymidine 5′-phosphorothioate.

TABLE 1 Beta-ENaC sequences Duplex SEQ SEQ ID ID Modified sequence IDUnmodified sequence Pos'n AD- S   1 cAGuGAcuAcAAcAcGAcc 111CAGUGACUACAACACGACC 1298 20805 AS   2 GGUCGUGUUGuAGUcACUG 112GGUCGUGUUGUAGUCACUG 1298 AD- S   3 AuGAcAGAGAAGGcAcuuc 113AUGACAGAGAAGGCACUUC 1011 20806 AS   4 GAAGUGCCUUCUCUGUcAU 114GAAGUGCCUUCUCUGUCAU 1011 AD- S   5 GuGAAGAAGuAccuGcuGA 115GUGAAGAAGUACCUGCUGA  183 20807 AS   6 UcAGcAGGuACUUCUUcAC 116UCAGCAGGUACUUCUUCAC  183 AD- S   7 GuGAcuAcAAcAcGAccuA 117GUGACUACAACACGACCUA 1300 20808 AS   8 uAGGUCGUGUUGuAGUcAC 118UAGGUCGUGUUGUAGUCAC 1300 AD- S   9 GGuGGAGGcccAcAccAAc 119GGUGGAGGCCCACACCAAC 1919 20809 AS  10 GUUGGUGUGGGCCUCcACC 120GUUGGUGUGGGCCUCCACC 1919 AD- S  11 uGGuGGAGGcccAcAccAA 121UGGUGGAGGCCCACACCAA 1918 20810 AS  12 UUGGUGUGGGCCUCcACcA 122UUGGUGUGGGCCUCCACCA 1918 AD- S  13 uuccAAGAccAcAuGAucc 123UUCCAAGACCACAUGAUCC 1347 20811 AS  14 GGAUcAUGUGGUCUUGGAA 124GGAUCAUGUGGUCUUGGAA 1347 AD- S  15 AGcuGGGAGGucAGcGucu 125AGCUGGGAGGUCAGCGUCU  402 20812 AS  16 AGACGCUGACCUCCcAGCU 126AGACGCUGACCUCCCAGCU  402 AD- S  17 GGGAGAAAuAcuGcAAcAA 127GGGAGAAAUACUGCAACAA 1408 20813 AS  18 UUGUUGcAGuAUUUCUCCC 128UUGUUGCAGUAUUUCUCCC 1408 AD- S  19 ccAGuuuGGcuucuGGAuG 129CCAGUUUGGCUUCUGGAUG 1748 20814 AS  20 cAUCcAGAAGCcAAACUGG 130CAUCCAGAAGCCAAACUGG 1748 AD- S  21 AGuGAcuAcAAcAcGAccu 131AGUGACUACAACACGACCU 1299 20815 AS  22 AGGUCGUGUUGuAGUcACU 132AGGUCGUGUUGUAGUCACU 1299 AD- S  23 AAuAucAcccuGAGcAGGA 133AAUAUCACCCUGAGCAGGA 1626 20816 AS  24 UCCUGCUcAGGGUGAuAUU 134UCCUGCUCAGGGUGAUAUU 1626 AD- S  25 ccuGcAGGccAccAAcAuc 135CCUGCAGGCCACCAACAUC  836 20817 AS  26 GAUGUUGGUGGCCUGcAGG 136GAUGUUGGUGGCCUGCAGG  836 AD- S  27 AucAcccuGAGcAGGAAGG 137AUCACCCUGAGCAGGAAGG 1629 20818 AS  28 CCUUCCUGCUcAGGGUGAU 138CCUUCCUGCUCAGGGUGAU 1629 AD- S  29 GcuGGGAGGucAGcGucuc 139GCUGGGAGGUCAGCGUCUC  403 20819 AS  30 GAGACGCUGACCUCCcAGC 140GAGACGCUGACCUCCCAGC  403 AD- S  31 GAGcuGGGAGGucAGcGuc 141GAGCUGGGAGGUCAGCGUC  401 20820 AS  32 GACGCUGACCUCCcAGCUC 142GACGCUGACCUCCCAGCUC  401 AD- S  33 GuGGccAGuuuGGcuucuG 143GUGGCCAGUUUGGCUUCUG 1744 20821 AS  34 cAGAAGCcAAACUGGCcAC 144CAGAAGCCAAACUGGCCAC 1744 AD- S  35 cAGuuuGGcuucuGGAuGG 145CAGUUUGGCUUCUGGAUGG 1749 20822 AS  36 CcAUCcAGAAGCcAAACUG 146CCAUCCAGAAGCCAAACUG 1749 AD- S  37 GGccAGuuuGGcuucuGGA 147GGCCAGUUUGGCUUCUGGA 1746 20823 AS  38 UCcAGAAGCcAAACUGGCC 148UCCAGAAGCCAAACUGGCC 1746 AD- S  39 cuGGGuGGccAGuuuGGcu 149CUGGGUGGCCAGUUUGGCU 1740 20824 AS  40 AGCcAAACUGGCcACCcAG 150AGCCAAACUGGCCACCCAG 1740 AD- S  41 ucuAcAGuGAcuAcAAcAc 151UCUACAGUGACUACAACAC 1294 20825 AS  42 GUGUUGuAGUcACUGuAGA 152GUGUUGUAGUCACUGUAGA 1294 AD- S  43 GcAuGAcAGAGAAGGcAcu 153GCAUGACAGAGAAGGCACU 1009 20826 AS  44 AGUGCCUUCUCUGUcAUGC 154AGUGCCUUCUCUGUCAUGC 1009 AD- S  45 AuAucAcccuGAGcAGGAA 155AUAUCACCCUGAGCAGGAA 1627 20827 AS  46 UUCCUGCUcAGGGUGAuAU 156UUCCUGCUCAGGGUGAUAU 1627 AD- S  47 cuAcAGuGAcuAcAAcAcG 157CUACAGUGACUACAACACG 1295 20828 AS  48 CGUGUUGuAGUcACUGuAG 158CGUGUUGUAGUCACUGUAG 1295 AD- S  49 uAucAcccuGAGcAGGAAG 159UAUCACCCUGAGCAGGAAG 1628 20829 AS  50 CUUCCUGCUcAGGGUGAuA 160CUUCCUGCUCAGGGUGAUA 1628 AD- S  51 uGcAGGccAccAAcAucuu 161UGCAGGCCACCAACAUCUU  838 20830 AS  52 AAGAUGUUGGUGGCCUGcA 162AAGAUGUUGGUGGCCUGCA  838 AD- S  53 cAuGAcAGAGAAGGcAcuu 163CAUGACAGAGAAGGCACUU 1010 20831 AS  54 AAGUGCCUUCUCUGUcAUG 164AAGUGCCUUCUCUGUCAUG 1010 AD- S  55 uGAAGAAGuAccuGcuGAA 165UGAAGAAGUACCUGCUGAA  184 20832 AS  56 UUcAGcAGGuACUUCUUcA 166UUCAGCAGGUACUUCUUCA  184 AD- S  57 GcuGGuGGAGGcccAcAcc 167GCUGGUGGAGGCCCACACC 1916 20833 AS  58 GGUGUGGGCCUCcACcAGC 168GGUGUGGGCCUCCACCAGC 1916 AD- S  59 uAcAGuGAcuAcAAcAcGA 169UACAGUGACUACAACACGA 1296 20834 AS  60 UCGUGUUGuAGUcACUGuA 170UCGUGUUGUAGUCACUGUA 1296 AD- S  61 AcAGAGAAGGcAcuuccuu 171ACAGAGAAGGCACUUCCUU 1014 20835 AS  62 AAGGAAGUGCCUUCUCUGU 172AAGGAAGUGCCUUCUCUGU 1014 AD- S  63 AcAGuGAcuAcAAcAcGAc 173ACAGUGACUACAACACGAC 1297 20836 AS  64 GUCGUGUUGuAGUcACUGU 174GUCGUGUUGUAGUCACUGU 1297 AD- S  65 uGAGcuGGGAGGucAGcGu 175UGAGCUGGGAGGUCAGCGU  400 20837 AS  66 ACGCUGACCUCCcAGCUcA 176ACGCUGACCUCCCAGCUCA  400 AD- S  67 uGGccAGuuuGGcuucuGG 177UGGCCAGUUUGGCUUCUGG 1745 20838 AS  68 CcAGAAGCcAAACUGGCcA 178CCAGAAGCCAAACUGGCCA 1745 AD- S  69 uGucucAGGAGcGGGAccA 179UGUCUCAGGAGCGGGACCA 1600 20839 AS  70 UGGUCCCGCUCCUGAGAcA 180UGGUCCCGCUCCUGAGACA 1600 AD- S  71 GuGGAGGcccAcAccAAcu 181GUGGAGGCCCACACCAACU 1920 20840 AS  72 AGUUGGUGUGGGCCUCcAC 182AGUUGGUGUGGGCCUCCAC 1920 AD- S  73 GGGuGGccAGuuuGGcuuc 183GGGUGGCCAGUUUGGCUUC 1742 20841 AS  74 GAAGCcAAACUGGCcACCC 184GAAGCCAAACUGGCCACCC 1742 AD- S  75 GGuGGccAGuuuGGcuucu 185GGUGGCCAGUUUGGCUUCU 1743 20842 AS  76 AGAAGCcAAACUGGCcACC 186AGAAGCCAAACUGGCCACC 1743 AD- S  77 ucAcccuGAGcAGGAAGGG 187UCACCCUGAGCAGGAAGGG 1630 20843 AS  78 CCCUUCCUGCUcAGGGUGA 188CCCUUCCUGCUCAGGGUGA 1630 AD- S  79 GccAGuuuGGcuucuGGAu 189GCCAGUUUGGCUUCUGGAU 1747 20844 AS  80 AUCcAGAAGCcAAACUGGC 190AUCCAGAAGCCAAACUGGC 1747 AD- S  81 AGcuGGuGGAGGcccAcAc 191AGCUGGUGGAGGCCCACAC 1915 20845 AS  82 GUGUGGGCCUCcACcAGCU 192GUGUGGGCCUCCACCAGCU 1915 AD- S  83 AucuccAuGGcuGAcuGGc 193AUCUCCAUGGCUGACUGGC 1545 20846 AS  84 GCcAGUcAGCcAUGGAGAU 194GCCAGUCAGCCAUGGAGAU 1545 AD- S  85 GGcAuGAcAGAGAAGGcAc 195GGCAUGACAGAGAAGGCAC 1008 20847 AS  86 GUGCCUUCUCUGUcAUGCC 196GUGCCUUCUCUGUCAUGCC 1008 AD- S  87 GGAGAAAuAcuGcAAcAAc 197GGAGAAAUACUGCAACAAC 1409 20848 AS  88 GUUGUUGcAGuAUUUCUCC 198GUUGUUGCAGUAUUUCUCC 1409 AD- S  89 uGGGuGGccAGuuuGGcuu 199UGGGUGGCCAGUUUGGCUU 1741 20849 AS  90 AAGCcAAACUGGCcACCcA 200AAGCCAAACUGGCCACCCA 1741 AD- S  91 GAGcuGGuGGAGGcccAcA 201GAGCUGGUGGAGGCCCACA 1914 20850 AS  92 UGUGGGCCUCcACcAGCUC 202UGUGGGCCUCCACCAGCUC 1914 AD- S  93 GAcAGAGAAGGcAcuuccu 203GACAGAGAAGGCACUUCCU 1013 20851 AS  94 AGGAAGUGCCUUCUCUGUC 204AGGAAGUGCCUUCUCUGUC 1013 AD- S  95 AGuuuGGcuucuGGAuGGG 205AGUUUGGCUUCUGGAUGGG 1750 20852 AS  96 CCcAUCcAGAAGCcAAACU 206CCCAUCCAGAAGCCAAACU 1750 AD- S  97 uGAcAGAGAAGGcAcuucc 207UGACAGAGAAGGCACUUCC 1012 20861 AS  98 GGAAGUGCCUUCUCUGUcA 208GGAAGUGCCUUCUCUGUCA 1012 AD- S  99 GAAGAAGuAccuGcuGAAG 209GAAGAAGUACCUGCUGAAG  185 20862 AS 100 CUUcAGcAGGuACUUCUUC 210CUUCAGCAGGUACUUCUUC  185 AD- S 101 ucuccAuGGcuGAcuGGcc 211UCUCCAUGGCUGACUGGCC 1546 20863 AS 102 GGCcAGUcAGCcAUGGAGA 212GGCCAGUCAGCCAUGGAGA 1546 AD- S 103 cuGGuGGAGGcccAcAccA 213CUGGUGGAGGCCCACACCA 1917 20864 AS 104 UGGUGUGGGCCUCcACcAG 214UGGUGUGGGCCUCCACCAG 1917 AD- S 105 cAGAGAAGGcAcuuccuuc 215CAGAGAAGGCACUUCCUUC 1015 20865 AS 106 GAAGGAAGUGCCUUCUCUG 216GAAGGAAGUGCCUUCUCUG 1015 AD- S 107 cuGcAGGccAccAAcAucu 217CUGCAGGCCACCAACAUCU  837 20866 AS 108 AGAUGUUGGUGGCCUGcAG 218AGAUGUUGGUGGCCUGCAG  837 AD- S 109 GGGcAuGAcAGAGAAGGcA 219GGGCAUGACAGAGAAGGCA 1007 20867 AS 110 UGCCUUCUCUGUcAUGCCC 220UGCCUUCUCUGUCAUGCCC 1007

Modifications of the sequences of RNAi agents of SEQ ID NO: 111 to 220are easily conceived by one of skill in the art. Examples andnon-limiting modifications of these sequences are conceived and are alsolisted in Table 1, e.g., the sense and antisense (AS) sequences in SEQID NO: 1 to 110.

Some modifications are placed at sites predicted to be sensitive toendonucleases. Some modifications are designed to eliminate an immuneresponse to the siRNA while preserving activity. In general, the sensestrand is heavily modified, and the antisense strand lightly modified.Some modifications serve more than one purpose.

The sequences in Table 1 and other tables are represented by theseabbreviations:

TABLE 1A ABBREVIATIONS Abbreviation Nucleotide(s) Aadenosine-5′-phosphate C cytidine-5′-phosphate G guanosine-5′-phosphatedT 2′-deoxy-thymidine-5′-phosphate U uridine-5′-phosphate c2′-O-methylcytidine-5′-phosphate u 2′-O-methyluridine-5′-phosphate sdT2′-deoxy Thymidine 5′-phosphorothioate

siRNA Sequence Selection

A total of 55 sense and 55 antisense human SCNNB1-derived siRNA oligos(RNAi agents to Beta-ENaC) are synthesized, as described in Example 2.The sense and their respective antisense oligos are annealed intoduplexes.

Example 1A Overlapping Sets of Beta-ENaC RNAi Agents

The present disclosure also relates to groups of RNAi agents toBeta-ENaC with overlapping sequences. Thus, the present disclosureencompasses groups of RNAi agents wherein each RNAi agent in the groupoverlaps with each other RNAi agent in the same group by at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides.Particularly, in one embodiment, the overlap is at least 12 nt.

Some of the RNAi agents listed herein overlap each other in sequence.Table 2 presents a compilation of some of these groups of overlappingRNAi agents, wherein each member of a group overlaps with each othermember of the same group by at least 12 nt. A 12-nt portion of theoverlap of the sense and anti-sense strand are presented.

Thus, for example, as shown in Table 2, the sequences of RNAi agentsAD-20807 and AD-20832 overlap, wherein the overlap in the sense strandcomprises the sequence UGAAGAAGUACC (SEQ ID NO: 223); these RNAi agentsalso overlap in the anti-sense strand sequence, wherein the overlapcomprises the sequence GGUACUUCUUCA (SEQ ID NO: 224). The RNAi agentsAD-20807, AD-20862 and AD-20832 all overlap in the sense strand, whereinthe overlap comprises the sequence GAAGAAGUACCU (SEQ ID NO: 225); theseRNAi agents also overlap in the anti-sense strand, wherein the overlapcomprises the sequence AGGUACUUCUUC (SEQ ID NO: 226). Thus, these andother various sets of overlapping RNAi agents presented in Table 2 sharecommon technical features, for example, the overlap in the sense andanti-sense strand.

Particular sets of overlapping RNAi agents to Beta-ENaC are providedbelow in Table 2.

The present disclosure thus encompasses any group or subgroup of RNAiagents comprising a common technical feature, wherein the commontechnical feature is an overlap (e.g., of at least 12 nt) of a sequencein the sense or anti-sense strand.

TABLE 2 Sense SEQ Anti-sense SEQ Overlapping RNAi Pos overlap ID overlapID agents to Beta-ENaC  183 UGAAGAAGUACC 223 GGUACUUCUUCA 224AD-20807, AD-20832  184 GAAGAAGUACCU 225 AGGUACUUCUUC 226AD-20807, AD-20862, AD-20832  185 AAGAAGUACCUG 227 CAGGUACUUCUU 228AD-20807, AD-20862, AD-20832  186 AGAAGUACCUGC 229 GCAGGUACUUCU 230AD-20807, AD-20862, AD-20832  187 GAAGUACCUGCU 231 AGCAGGUACUUC 232AD-20807, AD-20862, AD-20832  188 AAGUACCUGCUG 233 CAGCAGGUACUU 234AD-20807, AD-20862, AD-20832  189 AGUACCUGCUGA 235 UCAGCAGGUACU 236AD-20807, AD-20862, AD-20832  190 GUACCUGCUGAA 237 UUCAGCAGGUAC 238AD-20862, AD-20832  400 GAGCUGGGAGGU 239 ACCUCCCAGCUC 240AD-20820, AD-20837  401 AGCUGGGAGGUC 241 GACCUCCCAGCU 242AD-20820, AD-20812, AD-20837  402 GCUGGGAGGUCA 243 UGACCUCCCAGC 244AD-20820, AD-20819, AD-20812, AD-20837  403 CUGGGAGGUCAG 245CUGACCUCCCAG 246 AD-20819, AD-20812, AD-20837  404 UGGGAGGUCAGC 247GCUGACCUCCCA 248 AD-20820, AD-20819, AD-20837  405 GGGAGGUCAGCG 249CGCUGACCUCCC 250 AD-20820, AD-20819, AD-20812, AD-20837  406GGAGGUCAGCGU 251 ACGCUGACCUCC 252 AD-20819, AD-20837  407 GAGGUCAGCGUC253 GACGCUGACCUC 254 AD-20820, AD-20819, AD-20812  408 AGGUCAGCGUCU 255AGACGCUGACCU 256 AD-20819, AD-20812  836 CUGCAGGCCACC 257 GGUGGCCUGCAG258 AD-20866, AD-20817  837 UGCAGGCCACCA 259 UGGUGGCCUGCA 260AD-20866, AD-20830, AD-20817  838 GCAGGCCACCAA 261 UUGGUGGCCUGC 262AD-20866, AD-20830, AD-20817  839 CAGGCCACCAAC 263 GUUGGUGGCCUG 264AD-20866, AD-20830, AD-20817  840 AGGCCACCAACA 265 UGUUGGUGGCCU 266AD-20866, AD-20830, AD-20817  841 GGCCACCAACAU 267 AUGUUGGUGGCC 268AD-20866, AD-20830, AD-20817  842 GCCACCAACAUC 269 GAUGUUGGUGGC 270AD-20866, AD-20830, AD-20817  843 CCACCAACAUCU 271 AGAUGUUGGUGG 272AD-20866, AD-20830 1007 GGCAUGACAGAG 273 CUCUGUCAUGCC 274AD-20847, AD-20867 1008 GCAUGACAGAGA 275 UCUCUGUCAUGC 276AD-20826, AD-20867 1009 CAUGACAGAGAA 277 UUCUCUGUCAUG 278AD-20826, AD-20831, AD-20867 1010 AUGACAGAGAAG 279 CUUCUCUGUCAU 280AD-20826, AD-20831, AD-20867, AD-20806 1011 UGACAGAGAAGG 281CCUUCUCUGUCA 282 AD-20826, AD-20831, AD-20867, AD-20806, AD-20861 1012GACAGAGAAGGC 283 GCCUUCUCUGUC 284 AD-20851, AD-20847,AD-20826, AD-20831, AD-20867, AD-20806, AD-20861 1013 ACAGAGAAGGCA 285UGCCUUCUCUGU 286 AD-20851, AD-20835, AD-20847, AD-20826,AD-20831, AD-20867, AD-20806, AD-20861 1014 CAGAGAAGGCAC 287GUGCCUUCUCUG 288 AD-20851, AD-20835, AD-20865, AD-20826,AD-20831, AD-20806, AD-20861 1015 AGAGAAGGCACU 289 AGUGCCUUCUCU 290AD-20851, AD-20835, AD-20865, AD-20826, AD-20831, AD-20806, AD-208611016 GAGAAGGCACUU 291 AAGUGCCUUCUC 292 AD-20851, AD-20835,AD-20865, AD-20831, AD-20806, AD-20861 1017 AGAAGGCACUUC 293GAAGUGCCUUCU 294 AD-20851, AD-20835, AD-20865, AD-20806, AD-20861 1018GAAGGCACUUCC 295 GGAAGUGCCUUC 296 AD-20851, AD-20835, AD-20865, AD-208611019 AAGGCACUUCCU 297 AGGAAGUGCCUU 298 AD-20851, AD-20835, AD-20865 1020AGGCACUUCCUU 299 AAGGAAGUGCCU 300 AD-20835, AD-20865 1294 CUACAGUGACUA301 UAGUCACUGUAG 302 AD-20828, AD-20825 1295 UACAGUGACUAC 303GUAGUCACUGUA 304 AD-20834, AD-20825 1296 ACAGUGACUACA 305 UGUAGUCACUGU306 AD-20828, AD-20834, AD-20825, AD-20836 1297 CAGUGACUACAA 307UUGUAGUCACUG 308 AD-20834, AD-20805, AD-20825 1298 AGUGACUACAAC 309GUUGUAGUCACU 310 AD-20828, AD-20834, AD-20805, AD-20825, AD-20836 1299GUGACUACAACA 311 UGUUGUAGUCAC 312 AD-20834, AD-20805, AD-20808, AD-208251300 UGACUACAACAC 313 GUGUUGUAGUCA 314 AD-20828, AD-20834,AD-20805, AD-20808, AD-20825, AD-20815, AD-20836 1301 GACUACAACACG 315CGUGUUGUAGUC 316 AD-20828, AD-20834, AD-20805, AD-20808, AD-20836 1302ACUACAACACGA 317 UCGUGUUGUAGU 318 AD-20834, AD-20805, AD-20808 1303CUACAACACGAC 319 GUCGUGUUGUAG 320 AD-20805, AD-20808, AD-20815, AD-208361304 UACAACACGACC 321 GGUCGUGUUGUA 322 AD-20805, AD-20808 1305ACAACACGACCU 323 AGGUCGUGUUGU 324 AD-20808, AD-20815 1408 GGAGAAAUACUG325 CAGUAUUUCUCC 326 AD-20813, AD-20848 1409 GAGAAAUACUGC 327GCAGUAUUUCUC 328 AD-20813, AD-20848 1410 AGAAAUACUGCA 329 UGCAGUAUUUCU330 AD-20813, AD-20848 1411 GAAAUACUGCAA 331 UUGCAGUAUUUC 332AD-20813, AD-20848 1412 AAAUACUGCAAC 333 GUUGCAGUAUUU 334AD-20813, AD-20848 1413 AAUACUGCAACA 335 UGUUGCAGUAUU 336AD-20813, AD-20848 1414 AUACUGCAACAA 337 UUGUUGCAGUAU 338AD-20813, AD-20848 1545 UCUCCAUGGCUG 339 CAGCCAUGGAGA 340AD-20846, AD-20863 1546 CUCCAUGGCUGA 341 UCAGCCAUGGAG 342AD-20846, AD-20863 1547 UCCAUGGCUGAC 343 GUCAGCCAUGGA 344AD-20846, AD-20863 1548 CCAUGGCUGACU 345 AGUCAGCCAUGG 346AD-20846, AD-20863 1549 CAUGGCUGACUG 347 CAGUCAGCCAUG 348AD-20846, AD-20863 1550 AUGGCUGACUGG 349 CCAGUCAGCCAU 350AD-20846, AD-20863 1551 UGGCUGACUGGC 351 GCCAGUCAGCCA 352AD-20846, AD-20863 1626 AUAUCACCCUGA 353 UCAGGGUGAUAU 354AD-20816, AD-20827 1627 UAUCACCCUGAG 355 CUCAGGGUGAUA 356AD-20816, AD-20827, AD-20829 1628 AUCACCCUGAGC 357 GCUCAGGGUGAU 358AD-20816, AD-20827, AD-20829, AD-20818 1629 UCACCCUGAGCA 359UGCUCAGGGUGA 360 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1630CACCCUGAGCAG 361 CUGCUCAGGGUG 362 AD-20816, AD-20827,AD-20829, AD-20843, AD-20818 1631 ACCCUGAGCAGG 363 CCUGCUCAGGGU 364AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1632 CCCUGAGCAGGA 365UCCUGCUCAGGG 366 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1633CCUGAGCAGGAA 367 UUCCUGCUCAGG 368 AD-20827, AD-20829, AD-20843, AD-208181634 CUGAGCAGGAAG3 69 CUUCCUGCUCAG 370 AD-20829, AD-20843, AD-20818 1635UGAGCAGGAAGG 371 CCUUCCUGCUCA 372 AD-20843, AD-20818 1740 UGGGUGGCCAGU373 ACUGGCCACCCA 374 AD-20824, AD-20849 1741 GGGUGGCCAGUU 375AACUGGCCACCC 376 AD-20824, AD-20841, AD-20849 1742 GGUGGCCAGUUU 377AAACUGGCCACC 378 AD-20824, AD-20842, AD-20841, AD-20849 1743GUGGCCAGUUUG 379 CAAACUGGCCAC 380 AD-20824, AD-20842,AD-20821, AD-20841, AD-20849 1744 UGGCCAGUUUGG 381 CCAAACUGGCCA 382AD-20824, AD-20842, AD-20821, AD-20838, AD-20841, AD-20849 1745GGCCAGUUUGGC 383 GCCAAACUGGCC 384 AD-20824, AD-20842,AD-20821, AD-20838, AD-20841, AD-20823, AD-20849 1746 GCCAGUUUGGCU 385AGCCAAACUGGC 386 AD-20844, AD-20824, AD-20842, AD-20821,AD-20838, AD-20841, AD-20823, AD-20849 1747 CCAGUUUGGCUU 387AAGCCAAACUGG 388 AD-20814, AD-20844, AD-20842, AD-20821,AD-20838, AD-20841, AD-20823, AD-20849 1748 CAGUUUGGCUUC 389GAAGCCAAACUG 390 AD-20814, AD-20844, AD-20842, AD-20821,AD-20838, AD-20841, AD-20822, AD-20823 1749 AGUUUGGCUUCU 391AGAAGCCAAACU 392 AD-20814, AD-20844, AD-20842, AD-20821,AD-20852, AD-20838, AD-20822, AD-20823 1750 GUUUGGCUUCUG 393CAGAAGCCAAAC 394 AD-20814, AD-20844, AD-20821, AD-20852,AD-20838, AD-20822, AD-20823 1751 UUUGGCUUCUGG 395 CCAGAAGCCAAA 396AD-20814, AD-20844, AD-20852, AD-20838, AD-20822, AD-20823 1752UUGGCUUCUGGA 397 UCCAGAAGCCAA 398 AD-20814, AD-20844,AD-20852, AD-20822, AD-20823 1753 UGGCUUCUGGAU 399 AUCCAGAAGCCA 400AD-20814, AD-20844, AD-20852, AD-20822 1754 GGCUUCUGGAUG 401CAUCCAGAAGCC 402 AD-20814, AD-20852, AD-20822 1755 GCUUCUGGAUGG 403CCAUCCAGAAGC 404 AD-20852, AD-20822 1914 AGCUGGUGGAGG 405 CCUCCACCAGCU406 AD-20850, AD-20845 1915 GCUGGUGGAGGC 407 GCCUCCACCAGC 408AD-20850, AD-20845, AD-20833 1916 CUGGUGGAGGCC 409 GGCCUCCACCAG 410AD-20850, AD-20845, AD-20833, AD-20864 1917 UGGUGGAGGCCC 411GGGCCUCCACCA 412 AD-20810, AD-20850, AD-20845, AD-20833, AD-20864 1918GGUGGAGGCCCA 413 UGGGCCUCCACC 414 AD-20809, AD-20810,AD-20850, AD-20845, AD-20833, AD-20864 1919 GUGGAGGCCCAC 415GUGGGCCUCCAC 416 AD-20809, AD-20810, AD-20850, AD-20845,AD-20833, AD-20864, AD-20840 1920 UGGAGGCCCACA 417 UGUGGGCCUCCA 418AD-20809, AD-20810, AD-20850, AD-20845, AD-20833, AD-20864, AD-208401921 GGAGGCCCACAC 419 GUGUGGGCCUCC 420 AD-20809, AD-20810,AD-20845, AD-20833, AD-20864, AD-20840 1922 GAGGCCCACACC 421GGUGUGGGCCUC 422 AD-20809, AD-20810, AD-20833, AD-20864, AD-20840 1923AGGCCCACACCA 423 UGGUGUGGGCCU 424 AD-20809, AD-20810, AD-20864, AD-208401924 GGCCCACACCAA 425 UUGGUGUGGGCC 426 AD-20809, AD-20810, AD-20840 1925GCCCACACCAAC 427 GUUGGUGUGGGC 428 AD-20809, AD-20840

The position (“Pos”) in NM_(—)000336.2 is indicated. 12 exemplary nt ofthe overlap in the sense and anti-sense strand are presented; in manycases, the overlap is actually longer.

Example 2 Synthesis of Beta-ENaC RNAi Agent Sequences

The modified Beta-ENaC RNAi agent sequences listed as SEQ ID NO: 1 to110 in Table 1 are synthesized on MerMade 192 synthesizer at 1 μmolscale.

For all the sequences in the list, ‘endolight’ chemistry is applied asdetailed below.

All pyrimidines (cytosine and uridine) in the sense strand contain2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U).

In the antisense strand, pyrimidines adjacent to (i.e., towards the 5′position) ribo

A nucleoside are replaced with their corresponding 2-O-Methylnucleosides.

A two-base dTsdT extension at 3′ end of both sense and anti sensesequences is introduced.

The sequence file is converted to a text file to make it compatible forloading in the MerMade 192 synthesis software.

Synthesis, Cleavage and Deprotection:

The synthesis of Beta-ENaC sequences uses solid supportedoligonucleotide synthesis using phosphoramidite chemistry.

The synthesis of the above sequences is performed at 1 um scale in 96well plates. The amidite solutions are prepared at 0.1M concentrationand ethyl thio tetrazole (0.6M in Acetonitrile) is used as activator.

The synthesized sequences are cleaved and de-protected in 96 wellplates, using methylamine in the first step and fluoride reagent in thesecond step. The crude sequences are precipitated using acetone:ethanol(80:20) mix and the pellets are re-suspended in 0.2M sodium acetatebuffer. Samples from each sequence are analyzed by LC-MS to confirm theidentity, UV for quantification and a selected set of samples by IEXchromatography to determine purity.

Purification and Desalting:

Beta-ENaC sequences are purified on AKTA explorer purification systemusing Source 15Q column. A column temperature of 65 C is maintainedduring purification. Sample injection and collection is performed in 96well (1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence is collected in the eluent. The purified sequences aredesalted on a Sephadex G25 column using AKTA purifier. The desaltedBeta-ENaC sequences are analyzed for concentration (by UV measurement atA260) and purity (by ion exchange HPLC).

The single strands are then submitted for annealing.

A detailed list of Beta-ENaC single strands and duplexes are shown inTable 1, above. The duplexes are used in in vitro screening to testtheir ability to knock down Beta-ENaC gene level.

Example 3 In Vitro Screening of Beta-ENaC RNAi Agents

The Beta-ENaC RNAi agents are screened in vitro to determine theirability to knock down Beta-ENaC gene level.

Cell Culture and Transfections:

H441 (ATCC, Manassas, Va.) cells are grown to near confluence at 37° C.in an atmosphere of 5% CO₂ in RPMI 1640 (ATCC) supplemented with 10%FBS, streptomycin, and glutamine (ATCC) before being released from theplate by trypsinization. Reverse transfection is carried out by adding 5μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well platealong with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax perwell (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing 2.0×10⁴ H441 cells are then added. Cells areincubated for 24 hours prior to RNA purification. Experiments areperformed at 0.1 or 10 nM final duplex concentration for single dosescreens with each of the 55 Beta-ENaC duplexes. Each siRNA istransfected 3 times at each of the doses tested. The results are shownin Table 3.

A subset of duplexes that shows robust silencing in the 10 nM and 0.1 nMscreens is assayed over a range of concentrations from 10 nM to 10 fMusing serial dilutions to determine their IC50. The results are shown inTable 4.

Total RNA Isolation:

Cells are harvested and lysed in 140 μl of Lysis/Binding Solution thenmixed for 1 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process).

A MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Foster CityCalif., part #: AM1830) is used to isolate total RNA. Twenty microliters of magnetic beads and Lysis/Binding Enhancer mixture are addedinto cell-lysate and mixed for 5 minutes. Magnetic beads are capturedusing magnetic stand and the supernatant is removed without disturbingthe beads. After removing supernatant, magnetic beads are washed withWash Solution 1 (isopropanol added) and mixed for 1 minute. Beads arecapture again and supernatant removed. Beads are then washed with 150 μlWash Solution 2 (Ethanol added), captured and supernatant is removed. 50ul of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) is thenadded to the beads and they are mixed for 10 to 15 minutes. Aftermixing, 100 μl of RNA Rebinding Solution is added and mixed for 3minutes. Supernatant is removed and magnetic beads are washed again with150 μl Wash Solution 2 and mixed for 1 minute and supernatant is removedcompletely. The magnetic beads are mixed for 2 minutes to dry before RNAis eluted with 50 μl of water.

cDNA Synthesis:

ABI High capacity cDNA reverse transcription kit (Applied Biosystems,Foster City, Calif., Cat #4368813) is used for cDNA synthesis. A mastermix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μlReverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction are added into 10 μl total RNA. cDNA is generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA are added to a master mix containing 0.5 ul GAPDH TaqManProbe (Applied Biosystems. Cat #4326317E), 0.5 μl Beta-ENaC TaqMan probe(Applied Biosystems cat #Hs00165722_ml) and 5 μl Roche Probes Master Mix(Roche Cat #04887301001) in a total of 10 μl per well in a LightCycler480 384 well plate (Roche cat #0472974001). Real time PCR is done in aLightCycler 480 Real Time PCR machine (Roche). Each duplex is tested intwo independent transfections and each transfections is assayed induplicate.

Real time data are analyzed using the ΔΔCt method. Each sample isnormalized to GAPDH expression and knockdown is assessed relative tocells transfected with the non-targeting duplex AD-1955. IC50s aredefined using a 4 parameter fit model in XLfit.

The results are shown below. Table 3 shows the results of experimentsperformed at 0.1 nM or 10 nM final duplex concentrations for single dosescreens with each of the 55 Beta-ENaC duplexes. The “Fraction messageremaining” indicates the residual gene level, at 10 nm or 0.1 nM. Thus“0.17” in the second column for AD-20832-b1 indicates that, at aconcentration of 10 nM, there was 17% residual gene level, or 83%knockdown of expression. Note also that the suffix “b1” indicates “batch1.” Thus, for example, a RNAi agent with the designation “AD-20832-b1”has the same sequence as a RNAi agent designated “AD-20832”.

TABLE 3 10 nM and 0.1 nM knockdown of Beta-ENaC Fraction Fractionmessage message Standard Standard remaining remaining deviationdeviation At 10 nM At 0.1 nM At 10 nM At 0.1 nM AD-20832-b1 0.17 0.330.04 0.03 AD-20848-b1 0.17 0.49 0.01 0.04 AD-20807-b1 0.18 0.26 0.020.05 AD-20826-b1 0.19 0.49 0.02 0.22 AD-20837-b1 0.19 0.51 0.04 0.04AD-20861-b1 0.19 0.71 0.02 0.29 AD-20834-b1 0.20 0.34 0.06 0.05AD-20806-b1 0.22 0.60 0.02 0.15 AD-20851-b1 0.23 0.55 0.04 0.07AD-20865-b1 0.24 0.64 0.02 0.05 AD-20811-b1 0.25 0.52 0.17 0.23AD-20819-b1 0.27 0.60 0.01 0.07 AD-20839-b1 0.27 0.55 0.06 0.05AD-20835-b1 0.28 0.63 0.07 0.21 AD-20825-b1 0.30 0.72 0.11 0.15AD-20867-b1 0.30 0.68 0.00 0.20 AD-20813-b1 0.34 0.56 0.17 0.36AD-20823-b1 0.34 0.75 0.05 0.05 AD-20805-b1 0.36 0.86 0.02 0.09AD-20831-b1 0.36 0.60 0.01 0.21 AD-20862-b1 0.38 0.93 0.02 0.29AD-20808-b1 0.40 0.81 0.13 0.16 AD-20827-b1 0.40 2.55 0.07 1.44AD-20828-b1 0.42 0.89 0.11 0.25 AD-20812-b1 0.47 0.74 0.32 0.36AD-20836-b1 0.48 1.07 0.11 0.27 AD-20822-b1 0.49 0.94 0.11 0.09AD-20810-b1 0.53 0.87 0.25 0.20 AD-20824-b1 0.54 1.12 0.08 0.33AD-20844-b1 0.55 0.98 0.07 0.28 AD-20814-b1 0.60 1.30 0.09 0.12AD-20838-b1 0.65 1.18 0.07 0.18 AD-20816-b1 0.66 1.38 0.05 0.17AD-20845-b1 0.72 1.18 0.01 0.27 AD-20820-b1 0.75 0.89 0.06 0.14AD-20830-b1 0.75 0.94 0.04 0.24 AD-20866-b1 0.77 1.24 0.03 0.57AD-20809-b1 0.78 1.05 0.05 0.03 AD-20833-b1 0.79 0.99 0.01 0.35AD-20821-b1 0.80 0.99 0.07 0.14 AD-20846-b1 0.83 1.13 0.10 0.15AD-20818-b1 0.88 1.36 0.04 0.62 AD-20817-b1 0.89 1.11 0.11 0.19AD-20843-b1 0.92 1.64 0.11 0.16 AD-20840-b1 0.93 1.13 0.15 0.30AD-20847-b1 0.94 0.99 0.64 0.12 AD-20815-b1 0.96 2.06 0.23 0.99AD-20842-b1 0.96 1.37 0.16 0.28 AD-20852-b1 0.96 1.30 0.17 0.17AD-20863-b1 0.99 0.84 0.24 0.11 AD-20864-b1 0.99 1.36 0.05 0.74AD-20850-b1 1.00 1.22 0.14 0.14 AD-20829-b1 1.08 1.39 0.26 0.70AD-20849-b1 1.11 1.31 0.27 0.17 AD-20841-b1 1.12 1.37 0.10 0.48

All the RNAi agents to Beta-ENaC used in these experiments were themodified sequences (SEQ ID NO: 1 to 110) listed in Table 1.

Table 4 shows the results of experiments wherein a subset of duplexesthat show robust silencing in the 10 nM and 0.1 nM screens is assayedover a range of concentrations from 10 nM to 10 fM using serialdilutions to determine their IC50.

TABLE 4 Beta-ENaC dose response screen H441 New (Average H441 Old(Average of 4 replicates) of 8 replicates) IC50 IC50 IC50 Standard IC50Standard Duplex_ID nM deviation nM deviation AD-20807 0.05 0.03 0.040.06 AD-20826 0.14 0.05 0.05 0.07 AD-20832 0.05 0.02 0.04 0.05 AD-208340.06 0.03 0.03 0.06 AD-20848 0.25 0.14 0.13 0.17 AD-20861 0.13 0.08 0.090.06

Example 4 In Vivo Analysis of Beta-ENaC RNAi Agents AD-20807 andAD-20832

In in vivo experiments, two Beta-ENaC RNAi agents, AD-20807 andAD-20832, are tested for the ability to knock down Beta-ENaC gene levelin whole lungs in rats. The purpose is to determine the dose responses.Immunostimulation is also measured.

The Rat strain used is Sprague-Dawley; individuals have an approximateweight of 280-300 grams. Rats are dosed once a day for two days. Theyare then sacrificed about 24 hrs after the second dose. The left lung istaken and ground for qPCR determination of Beta-ENaC levels; the rightlung frozen and stored.

TABLE 5 Rat Rats per Group Numbers Formulation Concentration group 1 1-5D5W NA 5 2  6-10 AD1955 10 mg/kg 5 3 11-15 AD20191 10 mg/kg 5 4 16-20AD20807 10 mg/kg 5 5 12-25 AD20807  3 mg/kg 5 6 26-30 AD20807  1 mg/kg 57 31-35 AD20832 10 mg/kg  4* 8 36-40 AD20832  3 mg/kg 5 9 41-45 AD20832 1 mg/kg 5 *In the group of 4, 5 rats are initially dosed, but 1 in eachgroup does not survive the experiment and is not included in the finaldata.

Both RNAi agents to Beta-ENaC, AD20807 and AD20832, show reductions inBeta-ENaC levels in a dose-dependent manner. For AD20807, the level ofBeta-ENaC is reduced by approximately 30%, 40%, and 50% at dosages of 1,3 and 10 mg/kg, respectively.

In contrast, the Beta-ENaC (bENaC) RNAi agents do not decrease the levelof Alpha-ENaC (aENaC). There is, however, an increase in Alpha-ENaC withadministration of AD20832.

Negative Controls Include:

D5W: a solution of 5% dextrose in water; it is the vehicle used todilute the siRNA when dosing; AD1955: a siRNA which does notspecifically target either Alpha- or Beta-ENaC, but targets fireflyluciferase; and AD20191: a siRNA which does not bind to Beta-ENaC, buttargets rat Alpha-ENaC; and AD-9201, which targets Alpha-ENaC (not usedin this particular example).

Thus, specific knock-down of Beta-ENaC is seen with RNAi agent AD20807and AD20832 in this experiment.

Example 5 In Vivo Analysis of Beta-ENaC AD-20834

In in vivo experiments, Beta-ENaC RNAi agent AD20834 is tested for itsability to knock down Beta-ENaC gene level in whole lungs in rats. Thepurpose is to determine the dosage responses. Immunostimulation is alsomeasured.

The rat strain is Sprague-Dawley; individuals have an approximate weightof 280-300 grams. Rats are dosed once a day for two days. They are thensacrificed about 24 hours after the second dose. The left lung is takenand ground for qPCR determination of Beta-ENaC levels; the right lung isfrozen and stored.

TABLE 6 Group rat #s Formulation concentration rats per group 1 1-5 D5WNA 5 2  6-10 AD1955 10 mg/kg  4* 3 11-15 AD20191 10 mg/kg 5 4 16-20AD20834 10 mg/kg 5 5 21-25 AD20834  3 mg/kg 5 6 26-30 AD20834  1 mg/kg 4* *In the groups of 4, 5 rats are initially dosed, but 1 in each groupdoes not survive the experiment and is not included in the final data.

Assuming a weight of 300 grams (0.3 kg) the following dilutions aremade:

-   10 mg/kg=3 mg of siRNA in a 200 uL volume=15 mg/mL-   3 mg/kg=1 mg of siRNA in a 200 uL volume=5 mg/mL-   1 mg/kg=0.3 mg of siRNA in a 200 uL volume=1.5 mg/mL

The data are normalized to PPIB [Peptidyl-prolyl cis-trans isomerase B,used as a housekeeping (normalization) gene].

The experiments show that Beta-ENaC RNAi agent AD20834 demonstrates anapproximately 40% reduction in Beta-ENaC level in Sprague-Dawley rats.This effect is specific to Beta-ENaC.

The controls are as follows: D5W (5% dextrose in water) is a negativecontrol, not showing an effect on Alpha-ENaC or Beta-ENaC levels.AD1955, a control siRNA which does not bind to Alpha- or Beta-ENaC, alsoshowing little effect on Alpha- or Beta-ENaC level. The positive controlsiRNA AD20191, which targets Alpha- but not Beta-ENaC, demonstrates anapproximately 50% reduction in Alpha-ENaC level, but not Beta-ENaC.

Thus, a dosage of 10 mg/kg of Beta-ENaC RNAi agent AD20834 demonstratesat least about 40% inhibition of Beta-ENaC gene expression inSprague-Dawley rats.

Example 6 Analysis of Beta-ENaC RNAi Agents

Additional experimentation is done with Beta-ENaC RNAi agents AD20807,AD20832, AD20834, AD20848, and AD20861 in vivo in Sprague-Dawley rats.Rats are dosed at 10 mg/kg in D5W on day 1 and day 2, and are sacrificedon day 3, and the lungs are collected.

The results are shown in FIG. 1. The results show qPCR data from theleft lung, normalized to the control gene PPIB.

The controls in FIG. 1 are as follows: D5W (5% dextrose in water) is anegative control, not showing an effect on Alpha-ENaC or Beta-ENaClevels. The positive control is AD-9201, which targets Alpha-ENaC(aENaC).

The results, shown in FIG. 1, show a statistically significant andspecific knock-down of Beta-ENaC (bENaC) by AD20807, AD20832, AD20834,AD20848, and AD20861. The expression of the Beta-ENaC gene is inhibitedby at least about 40% at a concentration of 10 mg/kg of these RNAiagents in Sprague-Dawley rats.

Example 7 In Vitro Effect of Beta-ENaC RNAi Agent AD20832 on ENaCChannel Functional Activity in Human Bronchial Epithelial Cells

Human Bronchial Epithelial Cells (HBEC) are transfected with theindicated siRNA, including Beta-ENaC RNAi agent AD20832. Transfectedcells are seeded onto Snapwell inserts and cultured for 24 hours.Subsequently, the apical culture medium is removed from each insert andthe cells cultured at Air Liquid Interface (ALI). Cells are assayed forENaC and CFTR activity at Day 8 post-ALI as described. To control forcell viability, ENaC function is normalized to CFTR activity and thedata presented as a percentage relative to the untransfected control(FIG. 2A). As an additional viability control, trans-membrane resistanceis also measured (FIG. 2B). Expression analysis of alpha and beta ENaCsubunit mRNA is performed for each insert and normalized to GAPDHexpression. (FIG. 2C). The data demonstrate that a 70% inhibition ofmRNA expression is sufficient to generate a 50% functional inhibition ofENaC channel. This is true for knockdown of either alpha or betasubunits, where each is compared to untransfected (neg) and non-specific(ns) siRNA controls. The data also show that beta ENaC siRNA does notinhibit alpha ENaC mRNA expression and vice versa.

Methods: ENaC Functional Activity in Human Bronchial Epithelial Cells

Human Bronchial Epithelial Cells (HBEC) are purchased from Lonza andpassaged once before freezing in growth media (BEGM plussinglequots—Lonza). Subsequently, cells are thawed, expanded toconfluence and split 1:10 for transfection. Once at 80% confluence, eachflask of cells is transfected with the indicated siRNA at 30 nM, using 2μL/mL Lipofectamine 2000 in a total volume of 30 mL transfection media(1:1 mix of BEGM (Lonza) and DMEM high glucose (Gibco) with noadditives). At 24 hours post-transfection, cells are seeded onto 6 wellSnapwell inserts (Costar) at 2.5×10⁵ cells/insert in differentiationmedium (50:50 mix of BEBM and DMEM/high glucose with singlequots (minusthe tri-iodothreonine and retinoic acid supplements, with all-transretinoic acid added separately at 50 nM). Cells are supplemented with0.5 mL differentiation media apically and 2.5 mL differentiation mediabasolaterally. Following a further 24 hours of culture on the insertsthe basolateral media is replaced and the apical media is removed, thustaking the cells to Air Liquid Interface (ALI) culture. Cells areassayed for ENaC and CFTR activity at Day 8 (D8) Post-ALI.

To assess the ion transport phenotype of the transfected cells theSnapwell inserts are mounted in Vertical Diffusion Chambers (Costar) andare bathed continuously with gassed Ringer solution (5% CO₂ in O₂; pH7.4) maintained at 37° C. containing (in mM): 120 NaCl, 25 NaHCO₃, 3.3KH₂PO₄, 0.8 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, and 10 glucose (Osmolaritymaintained between 280 and 300 mosmol/l). Cells are voltage clamped to 0mV (model EVC4000; WPI). Trans-membrane resistance (TM res) is measuredby applying a 2-mV pulse at 30-s intervals and calculating TM res usingOhm's law. Short circuit current data are recorded using a PowerLabworkstation (ADI Instruments). Activity of the ENaC channel in eachgroup is assessed by the change in short-circuit current following theapical addition of 10 μM of the ENaC blocker Amiloride(Amiloride-sensitive current). Chloride secretion via CFTR is assessedby the change in short circuit current following apical and basolateraladdition of 0.6 μM Forskolin which is known to activate CFTR (Forskolinresponse). For each insert the Amiloride-sensitive current is normalizedto the Forskolin response and the data presented as a percentagerelative to the untransfected control. At the end of the study eachinsert is lysed for RNA analysis (300 μL RLT Buffer—Qiagen) and samplesretained for subsequent analysis of mRNA knockdown by rtPCR asdescribed.

Abbreviations:

-   ALI Air-Liquid Interface-   BEGM Bronchial Epithelial Growth Medium-   D6, D8 Day 6, Day 8-   DMEM Dulbecco's Modified Eagle Medium-   HBEC Human Bronchial Epithelial Cells-   TM res Trans-membrane resistance

EQUIVALENTS

A composition comprising a RNAi agent comprising a sense strand and anantisense strand, wherein the antisense strand comprises at least 15contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from theantisense strand of a RNAi agent specific to Beta-ENaC provided in Table1.

The composition of paragraph [00879], wherein the composition furthercomprises a second RNAi agent to Beta-ENaC.

The composition of paragraph [00879], wherein the antisense strand is 30or fewer nucleotides in length.

The composition of paragraph [00879], wherein the sense strand and theantisense strand form a duplex region 15 to 30 nucleotide pairs inlength.

The composition of paragraph [00879], wherein the antisense strand andthe sense strand are independently 19 to 23 nucleotides in length.

The composition of paragraph [00879], wherein the RNAi agent comprises amodification that causes the RNAi agent to have increased stability in abiological sample or environment.

The composition of paragraph [00879], wherein the RNAi agent comprisesat least one modified backbone and/or at least one 2′-modifiednucleotide.

The composition of paragraph [00879], wherein the RNAi agent comprises:

a) at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, whereinthe uridine is a 2′-modified nucleotide; and/or

b) at least one 5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, whereinthe 5′-uridine is a 2′-modified nucleotide; and/or

c) at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, whereinthe 5′-cytidine is a 2′-modified nucleotide; and/or

d) at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, whereinthe 5′-uridine is a 2′-modified nucleotide.

The composition of paragraph [00879], wherein the RNAi agent comprises a2′-modification selected from the group consisting of: 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O13 NMA).

The composition of cla paragraph [00879]1, wherein the RNAi agentcomprises a blunt end.

The composition of paragraph [00879], wherein the RNAi agent comprisesan overhang having 1 to 4 unpaired nucleotides.

The composition of paragraph [00879], wherein the RNAi agent comprisesan overhang at the 3′-end of the antisense strand of the RNAi agent.

The composition of paragraph [00879], wherein the RNAi agent is ligatedto one or more agent selected from: diagnostic compound, reporter group,cross-linking agent, nuclease-resistance conferring moiety, natural orunusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin,steroid, uvaol, hecigenin, diosgenin, terpene, triterpene,sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid,vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, syntheticcarbohydrate, oligo lactate 15-mer, natural polymer, low- ormedium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid,protein, protein-binding agent, integrin-targeting molecule,polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

The composition of paragraph [00879], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 60% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00879], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 70% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00879], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 80% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00879], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 90% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00879], wherein the RNAi has an EC50 of nomore than about 0.1 nM.

The composition of paragraph [00879], wherein the RNAi has an EC50 of nomore than about 0.01 nM.

The composition of paragraph [00879], wherein the RNAi has an EC50 of nomore than about 0.001 nM.

A composition comprising a RNAi agent comprising a first strand and asecond strand, wherein the first strand and second strand comprise atleast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotidesfrom the first and second strand, respectively, of a RNAi agent specificto Beta-ENaC provided in Table 1.

The composition of paragraph [00899], wherein the composition comprisesa second RNAi agent to Beta-ENaC.

The composition of paragraph [00899], wherein the second strand is 30 orfewer nucleotides in length.

The composition of paragraph [00899], wherein the first strand and thesecond strand form a duplex region 15 to 30 nucleotide pairs in length.

The composition of paragraph [00899], wherein the first strand and thesecond strand are independently 19 to 23 nucleotides in length.

The composition of paragraph [00899], wherein the RNAi agent comprises amodification that causes the RNAi agent to have increased stability in abiological sample or environment.

The composition of paragraph [00899], wherein the RNAi agent comprises aphosphorothioate and/or a 2′-modified nucleotide.

The composition of paragraph [00899], wherein the RNAi agent comprises:

at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein theuridine is a 2′-modified nucleotide; and/or at least one5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide;

and/or at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide,wherein the 5′-cytidine is a 2′-modified nucleotide;

and/or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide,wherein the 5′-uridine is a 2′-modified nucleotide.

The composition of paragraph [00899], wherein the RNAi agent comprisesone or more 2′-modifications selected from the group consisting of:

2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O—NMA).

The composition of paragraph [00899], wherein the RNAi agent comprises ablunt end.

The composition of paragraph [00899], wherein the RNAi agent comprisesan overhang having 1 to 4 unpaired nucleotides.

The composition of paragraph [00899], wherein the RNAi agent comprisesan overhang at the 3′-end of the antisense strand.

The composition of paragraph [00899], wherein the RNAi agent is ligatedto one or more agents, the agent selected from a: diagnostic compound,reporter group, cross-linking agent, nuclease-resistance conferringmoiety, natural or unusual nucleobase, lipophilic molecule, cholesterol,lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene,triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatizedlithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin,chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer,low- or medium-molecular weight polymer, inulin, cyclodextrin,hyaluronic acid, protein, protein-binding agent, integrin-targetingmolecule, polycationic, peptide, polyamine, peptide mimic, and/ortransferrin.

The composition of paragraph [00899], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 60% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00899], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 70% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00899], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 80% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00899], wherein the RNAi agent is capableof inhibiting expression of the Beta-ENaC gene by at least about 90% ata concentration of 10 nM in H441 cells in vitro.

The composition of paragraph [00899], wherein the RNAi has an EC50 of nomore than about 0.1 nM.

The composition of paragraph [00899], wherein the RNAi has an EC50 of nomore than about 0.01 nM.

The composition of paragraph [00899], wherein the RNAi has an EC50 of nomore than about 0.001 nM.

A method of treating a Beta-ENaC-related disease in an individual,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising a RNAi agent comprising asense strand and an antisense strand, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the antisense strand of a RNAi agent specific toBeta-ENaC provided in Table 1.

The method of paragraph [00919], wherein the Beta-ENaC-related diseaseis cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle'ssyndrome, hypertension, alkalosis, hypokalemia, and/orobesity-associated hypertension.

The method of paragraph [00919], wherein the Beta-ENaC-related diseaseis cystic fibrosis.

The method of paragraph [00919], wherein the method further comprisesthe step of administering an additional treatment for cystic fibrosis,pseudohypoaldosteronism type 1 (PHA1), Liddle's syndrome, hypertension,alkalosis, hypokalemia, and/or obesity-associated hypertension.

The method of paragraph [00919], wherein the composition comprises asecond RNAi agent to Beta-ENaC.

The method of paragraph [00919], wherein the method further comprisesthe step of administering an additional RNAi agent to Beta-ENaC.

The method of paragraph [00919], further comprising the administrationof an additional treatment.

The method of paragraph [00919], wherein the additional treatment is acomposition.

The method of paragraph [00919], wherein the additional treatment is amethod.

The method of paragraph [00919], wherein the additional treatment andthe RNAi agent can be administered in any order.

A method of inhibiting the expression of the Beta-ENaC gene in anindividual, comprising the step of administering to the individual atherapeutically effective amount of a composition comprising a RNAiagent comprising a sense strand and an antisense strand, wherein theantisense strand comprises at least 15 contiguous nucleotides differingby 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agentspecific to Beta-ENaC provided in Table 1.

The method of paragraph [00929], wherein the individual is afflictedwith or susceptible to a Beta-ENaC-related disease.

The method of paragraph [00929], wherein the Beta-ENaC-related diseaseis cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle'ssyndrome, hypertension, alkalosis, hypokalemia, and/orobesity-associated hypertension.

The method of paragraph [00929], wherein the Beta-ENaC-related diseaseis cystic fibrosis.

The method of paragraph [00932], further comprising the administrationof an additional treatment.

The method of paragraph [00933], wherein the additional treatment is acomposition.

The method of paragraph [00933], wherein the additional treatment is amethod.

The method of paragraph [00933], wherein the additional treatment andthe RNAi agent can be administered in any order.

A medicament for use in an RNAi formulation comprising a RNAi agentcomprising a sense strand and an antisense strand, wherein the antisensestrand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the antisense strand of a RNAi agent specificto Beta-ENaC provided in Table 1.

Any composition above in a pharmaceutically effective formulation.

The composition according to paragraph [00879], for use in a method oftreating a Beta-ENaC-related disease in an individual, the methodcomprising the step of administering to the individual a therapeuticallyeffective amount of a composition according to paragraph [00879].

The use of a composition according to paragraph [00879], in themanufacture of a medicament for the treatment of a Beta-ENaC-relateddisease.

The use of paragraph [00940], wherein the Beta-ENaC-related disease iscystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle'ssyndrome, hypertension, alkalosis, hypokalemia, and/orobesity-associated hypertension.

The composition of paragraph [00879], wherein all the pyrimidines are 2′O-methyl-modified nucleotides.

The composition of paragraph [00899], wherein all the pyrimidines are 2′O-methyl-modified nucleotides.

1. A composition comprising a first RNAi agent comprising a first strandand a second strand, wherein: a. the sequence of first strand comprisesat least 15 contiguous nucleotides differing by 0, 1, 2, or 3nucleotides from the sequence of SEQ ID NO: 154, or b. the sequence ofthe first strand comprises at least 15 contiguous nucleotides differingby 0, 1, 2 or 3 nucleotides from the sequence of SEQ ID NO: 208; whereinthe length of the first and second strand are each at least about 15nucleotides and no more than about 30 nucleotides, and wherein the firstand/or the second strand are modified or unmodified, and wherein thecomposition further comprises a second RNAi agent to Beta-ENaC.
 2. Thecomposition of claim 1, wherein the first and/or second RNAi agentcomprises at least one modified backbone and/or at least one 2′-modifiednucleotide.
 3. The composition of claim 1, wherein the first and/orsecond RNAi agent is ligated to one or more agents, wherein the one ormore agents are selected from: diagnostic compound, reporter group,cross-linking agent, nuclease-resistance conferring moiety, natural orunusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin,steroid, uvaol, hecigenin, diosgenin, terpene, triterpene,sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid,vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, syntheticcarbohydrate, oligo lactate 15-mer, natural polymer, low- ormedium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid,protein, protein-binding agent, integrin-targeting molecule,polycationic, peptide, polyamine, peptide mimic, and transferrin.
 4. Acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein: a. the sequence of the first strand comprisesthe sequence of SEQ ID NO: 154, or b. the sequence of the first strandcomprises the sequence of SEQ ID NO: 208; wherein the length of thefirst and second strand are each no more than about 30 nucleotides, andwherein the first and/or the second strand are modified or unmodified.5. The composition of claim 4, wherein the composition further comprisesa second RNAi agent to Beta-ENaC.
 6. The composition of claim 4, whereinthe RNAi agent comprises a phosphorothioate and/or a 2′-modifiednucleotide.
 7. The composition of claim 4, wherein the RNAi agent isligated to one or more agents, wherein the one or more agents areselected from: diagnostic compound, reporter group, cross-linking agent,nuclease-resistance conferring moiety, natural or unusual nucleobase,lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol,hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and transferrin.
 8. A method of reducing the expressionand/or level of Beta-ENaC in an individual, the method comprising thestep of administering to the individual a therapeutically effectiveamount of a composition comprising a pharmaceutically acceptable carrierand a RNAi agent comprising a first strand and a second strand, wherein:a. the sequence of the first strand comprises the sequence of SEQ ID NO:154, or b. the sequence of the first strand comprises the sequence ofSEQ ID NO: 208; wherein the length of the first and second strand areeach no more than about 30 nucleotides, and wherein the first and/or thesecond strand are modified or unmodified.
 9. The method of claim 8,wherein the method further comprises the step of administering anadditional RNAi agent to Beta-ENaC.
 10. A composition comprising a RNAiagent comprising a first strand and a second strand, wherein: a. thesequence of the first strand is the sequence of SEQ ID NO: 154, and/orthe sequence of the second strand is the sequence of SEQ ID NO: 153, orb. the sequence of the first strand is the sequence of SEQ ID NO: 208,and/or the sequence of the second strand is the sequence of SEQ ID NO:207; and wherein the first and/or the second strand are modified orunmodified.
 11. The composition of claim 10, wherein the RNAi agentcomprises one or more 2′-modifications selected from the groupconsisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAEO), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O—NMA).
 12. The composition of claim 10,wherein the RNAi agent is ligated to one or more agents, wherein the oneor more agents are selected from: diagnostic compound, reporter group,cross-linking agent, nuclease-resistance conferring moiety, natural orunusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin,steroid, uvaol, hecigenin, diosgenin, terpene, triterpene,sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid,vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, syntheticcarbohydrate, oligo lactate 15-mer, natural polymer, low- ormedium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid,protein, protein-binding agent, integrin-targeting molecule,polycationic, peptide, polyamine, peptide mimic, and transferrin. 13.The composition of claim 10, wherein the composition comprises a secondRNAi agent to Beta-ENaC.
 14. The composition of claim 10, wherein theRNAi agent comprises a phosphorothioate and/or a 2′-modified nucleotide.15. A composition comprising a therapeutically effective amount of acomposition of claim 4 and a pharmaceutically acceptable carrier.
 16. Acomposition comprising a therapeutically effective amount of acomposition of claim 10 and a pharmaceutically acceptable carrier.
 17. Acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein: a. the sequence of the first strand is thesequence of SEQ ID NO: 154, or b. the sequence of the first strand isthe sequence of SEQ ID NO: 208; and wherein the first and/or the secondstrand are modified or unmodified.